U.S. patent application number 15/897063 was filed with the patent office on 2018-11-15 for cd19 binding agents and uses thereof.
The applicant listed for this patent is SEATTLE GENETICS, INC.. Invention is credited to Dennis Benjamin, Paul Carter, Charles G. Cerveny, Charlotte McDonagh.
Application Number | 20180326087 15/897063 |
Document ID | / |
Family ID | 40568090 |
Filed Date | 2018-11-15 |
United States Patent
Application |
20180326087 |
Kind Code |
A1 |
McDonagh; Charlotte ; et
al. |
November 15, 2018 |
CD19 BINDING AGENTS AND USES THEREOF
Abstract
This invention, inter alia, relates to CD19 binding agents and
methods of using such CD19 binding agents for treating disease.
Inventors: |
McDonagh; Charlotte;
(Waltham, MA) ; Cerveny; Charles G.; (Seattle,
WA) ; Benjamin; Dennis; (Redmond, WA) ;
Carter; Paul; (Mercer Island, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SEATTLE GENETICS, INC. |
Bothell |
WA |
US |
|
|
Family ID: |
40568090 |
Appl. No.: |
15/897063 |
Filed: |
February 14, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14743318 |
Jun 18, 2015 |
9919061 |
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15897063 |
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13530074 |
Jun 21, 2012 |
9073993 |
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14743318 |
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13109957 |
May 17, 2011 |
8242252 |
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13530074 |
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12253895 |
Oct 17, 2008 |
7968687 |
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13109957 |
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60981206 |
Oct 19, 2007 |
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61019214 |
Jan 4, 2008 |
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61080169 |
Jul 11, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/6803 20170801;
C07K 2317/56 20130101; A61P 37/06 20180101; A61K 38/05 20130101;
C07K 16/2803 20130101; Y02A 50/423 20180101; C07K 2317/565
20130101; A61K 47/6811 20170801; Y02A 50/401 20180101; Y02A 50/412
20180101; C07K 2317/24 20130101; A61K 47/6867 20170801; Y02A 50/30
20180101; Y02A 50/414 20180101; C07K 16/3061 20130101; A61P 35/02
20180101; Y02A 50/41 20180101; A61P 37/02 20180101; C07K 2317/77
20130101; A61K 47/6849 20170801; A61K 2039/505 20130101; C07K
2317/732 20130101; A61P 35/00 20180101 |
International
Class: |
A61K 47/68 20060101
A61K047/68; A61K 38/05 20060101 A61K038/05; C07K 16/30 20060101
C07K016/30; C07K 16/28 20060101 C07K016/28 |
Claims
1-15. (canceled)
16. A CD19 binding agent that specifically binds to human CD19,
said binding agent comprising a heavy chain variable region
comprising an amino acid sequence at least 90% identical to SEQ ID
NO:2; the binding agent binding to human CD19 with a dissociation
constant of less than 10.sup.-7M.
17. The CD19 binding agent of claim 16 that specifically binds to
human CD19 and comprises a heavy chain variable region comprising
an amino acid sequence at least 90% identical to SEQ ID NO:2; the
binding agent binding to human CD19 with a dissociation constant of
less than 10.sup.-7M and comprising CDR regions having the amino
acid sequences set forth in SEQ ID NOS. 46, 47, and 48.
18. The binding agent of claim 16 further comprising a light chain
variable region comprising an amino acid sequence at least 90%
identical to SEQ ID NO:17 or SEQ ID NO:26.
19. The CD19 binding agent of claim 16, further comprising a human
IgG constant region joined to the heavy chain variable region.
20. The CD19 binding agent of claim 19, wherein the isotype of IgG
constant region is IgG1, IgG2, or IgG1V1.
21. The CD19 binding agent of claim 18 further comprising a light
chain constant domain joined to the light chain variable region,
wherein the light chain constant domain is a kappa constant
domain.
22. The CD19 binding agent of claim 16 that comprises a humanized
antibody.
23. The CD19 binding agent of claim 16 wherein the CD19 binding
agent is conjugated to a cytotoxic agent.
24. A ligand-drug conjugate compound of the following formula:
L-(LU-D).sub.p (I) or a pharmaceutically acceptable salt or solvate
thereof; wherein: L is a ligand unit wherein the ligand unit is a
CD19 binding agent of claim 16; and (LU-D) is a Linker unit-Drug
unit moiety, wherein: LU- is a Linker unit, and -D is a Drug unit
having cytostatic or cytotoxic activity against the target cells;
and p is an integer from 1 to about 20.
25. The ligand-drug conjugate compound of claim 24, wherein the
ligand-drug conjugate compound has the formula: ##STR00036## or a
pharmaceutically acceptable salt or solvate form thereof, wherein
mAb-S-- is a CD19 binding agent of claim 1 and p is from 1 to
8.
26. A ligand-drug conjugate compound having the formula:
##STR00037## or a pharmaceutically acceptable salt or solvate form
thereof; wherein mAb-S-- is a CD19 binding agent comprising a heavy
chain variable region comprising the amino acid sequence of SEQ ID
NO:9 and comprising a light chain variable region comprising the
amino acid sequence of SEQ ID NO:24 and p is from 1 to 8. or a
pharmaceutically acceptable salt or solvate form thereof.
27. A pharmaceutical composition comprising the ligand-drug
conjugate compound of claim 1, and a pharmaceutically acceptable
carrier or excipient.
28. A method for treating a CD19-associated disorder in a mammalian
subject comprising administering a pharmaceutical composition of
claim 27 in an amount effective to treat the disorder in the
mammalian subject.
29. The method of claim 28 wherein the CD19-associated disorder is
a CD19 expressing cancer.
30. A method for treating a subject that has a cancer that is
refractory to treatment with rituximab comprising administering to
the subject a pharmaceutical composition of claim 27 in an amount
effective to treat the cancer.
31. A method of manufacturing a ligand-drug conjugate compound
comprising conjugating a CD19 binding agent of claim 1 to a
cytotoxic agent.
32. A method of manufacturing a ligand-drug conjugate compound
comprising conjugating a CD19 binding agent of claim 1 to a linker
unit conjugated to a drug unit.
33. The binding agent of claim 3 comprising a humanized antibody
wherein the heavy chain variable region comprises an amino acid
sequence at least 90% identical to SEQ ID NO:2 and the light chain
variable region comprises an amino acid sequence at least 90%
identical to SEQ ID NO:17.
34. The humanized antibody of claim 33, wherein any heavy chain
variable region framework positions that differ from SEQ ID NO:2
are occupied by the amino acids occupying the corresponding
positions of SEQ ID NO:8 or SEQ ID NO:32; and wherein any light
chain variable region framework positions that differ from SEQ ID
NO:17 are occupied by the amino acids occupying the corresponding
position of SEQ ID NO:25 or SEQ ID NO:34.
35. The humanized antibody of claim 34, wherein any heavy chain
variable region framework positions that differ from SEQ ID NO:2
and the amino acids occupying those positions are selected from the
group consisting of positions H75, H79, H81, H82, H82A, H82B, H82C,
and H89 occupied by S, F, K, I, A, S, V, and A respectively; and
any light chain variable region framework positions that differ
from SEQ ID NO.17 and the amino acids occupying those positions are
selected from the group consisting of positions L2, L40, L41, L42,
L69, L70, L71, L72 and L83 occupied by N, S, S, T, N, S, H, F, and
V respectively.
36. The humanized antibody of claim 35, wherein the light chain
variable region framework position L83 is occupied by V.
37. The humanized antibody of claim 33, wherein at least one of the
heavy chain variable region positions H75, H79, H81, H82, H82A,
H82B, H82C and H89 is occupied by S, F, K, I, A, S, V, and A
respectively.
38. The humanized antibody of claim 33, wherein at least one of the
light chain variable region positions L2, L69, L71, L72 and L83 is
occupied by N, N, H, F and V respectively.
39. The humanized antibody of claim 33, wherein no more than three
amino acids in the heavy chain variable region framework differ
from SEQ ID NO:2, and no more than three amino acids in the light
chain variable region framework differ from SEQ ID NO:17.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 14/743,318 filed Jun. 18, 2015, which is a continuation of U.S.
application Ser. No. 13/530,074 filed Jun. 21, 2012, now U.S. Pat.
No. 9,073,993, which is a divisional of Ser. No. 13/109,957 filed
May 17, 2011, now U.S. Pat. No. 8,242,252, which is a divisional of
Ser. No. 12/253,895 filed Oct. 17, 2008, now U.S. Pat. No.
7,968,687, which claims the benefit of U.S. Provisional App. No.
60/981,206 filed Oct. 19, 2007; U.S. Provisional App. No.
60/019,214 filed Jan. 4, 2008; and U.S. Provisional App. No.
61/080,169 filed Jul. 11, 2008, each of which is incorporated by
reference in its entirety for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a sequence listing as a text file
named "510783_SEQLIST.txt" created on Feb. 13, 2018, and containing
73,727 bytes, which is incorporated by reference in its entirety
for all purposes.
FIELD
[0003] This invention relates to CD19 binding agents comprising a
humanized variable region and methods of using such CD19 binding
agents for treating disease characterized by expression of CD19
antigen.
BACKGROUND
[0004] In humans, B cells can produce an enormous number of
antibody molecules. Such antibody production typically ceases (or
substantially decreases) when a foreign antigen has been
neutralized. Occasionally, however, proliferation of a particular B
cell will continue unabated and can result in a cancer known as a B
cell lymphoma. B-cell lymphomas, such as the B-cell subtype of
non-Hodgkin lymphoma, are significant contributors to cancer
mortality. The response of B-cell malignancies to various forms of
treatment is mixed. Despite the medical importance, research in
B-cell mediated diseases such as non-Hodgkin lymphoma has produced
only a small number of clinically usable data and conventional
approaches to treat such diseases remain tedious and unpleasant
and/or have a high risk of relapse. For example, although high dose
chemotherapy as a primary treatment for high grade non-Hodgkin
lymphoma can improve overall survival, about 50% of the patients
still die of this disease. Devesa et al., J. Nat'l Cancer Inst. 79:
701 (1987). Moreover, low-grade non-Hodgkin lymphoma-like chronic
lymphocytic leukemia and mantle cell lymphoma are still incurable.
This has stimulated the search for alternative strategies like
immunotherapy. Antibodies directed against cell surface molecules
defined by CD antigens represent a unique opportunity for the
development of therapeutic reagents.
[0005] The majority of chronic lymphocytic leukemias are of the
B-cell lineage. Freedman, Hematol. Oncol. Clin. North Am. 4: 405,
1990. This type of B-cell malignancy is the most common leukemia in
the Western world. Goodman et al., Leukemia and Lymphoma 22: 1,
1996. The natural history of chronic lymphocytic leukemia falls
into several phases. In the early phase, chronic lymphocytic
leukemia is an indolent disease, characterized by the accumulation
of small mature functionally-incompetent malignant B-cells having a
lengthened life span. Eventually, the doubling time of the
malignant B-cells decreases and patients become increasingly
symptomatic. While treatment can provide symptomatic relief, the
overall survival of the patients is only minimally affected. The
late stages of chronic lymphocytic leukemia are characterized by
significant anemia and/or thrombocytopenia. At this point, the
median survival is less than two years. Foon et al., Annals Int.
Medicine 113: 525 (1990).
[0006] B cells express cell surface proteins which can be utilized
as markers for differentiation and identification. CD19 is a pan-B
cell membrane glycoprotein that is expressed from early stages of
pre-B cell development through terminal differentiation, regulating
B lymphocyte development and function. Expression of CD19 was
identified on most cancers of lymphoid origin, on the vast majority
of Non-Hodgkin lymphoma (NHL) and on leukemias, including Chronic
Lymphocytic Leukemia (CLL), Acute Lymphoblastic Leukemia (ALL) and
Waldenstrom's Macroglobulinemia (WM). Despite major improvements in
the treatment of NHL and CLL patients, the majority will continue
to relapse and salvage regimens with non-cross resistant compounds
are required to improve patient survival. A need exists in the art
for improved methods of treatment. The present invention addresses
this and other needs.
SUMMARY
[0007] The invention provides, inter alia, CD19 binding agents and
methods of using such binding agents. In some aspects, the binding
agents comprise the amino acid sequence(s) of a humanized heavy
chain variable region and/or a humanized light chain variable
region and specifically bind to human CD19. In some embodiments,
the CD19 binding agent is an antigen-binding antibody fragment that
specifically binds to human CD19. The antibody fragment can be, for
example, a Fab, Fab', F(ab').sub.2, Fv fragment, a diabody, a
linear antibody, an scFv, or an scFv-Fc.
[0008] In some aspects, the CD19 binding agent has a cytotoxic,
cytostatic and/or immunomodulatory effect on CD19-expressing cells.
Such an effect can be mediated, for example, by the depletion or
inhibition of the proliferation or differentiation of
CD19-expressing cells. In some embodiments, the CD19 binding agent
can mediate effector function. In some embodiments, the CD19
binding agent is conjugated to a therapeutic agent. In other
embodiments, the CD19 binding agent is unconjugated, i.e., not
conjugated to a therapeutic agent (for example, an anti-CD19 naked
antibody).
[0009] The present invention provides, inter alia, ligand-drug
conjugate compounds wherein the ligand unit is a CD19 binding agent
of the present invention. The ligand-drug conjugates can be used,
for example, to treat an immune disorder or cancer.
[0010] Cancers to be treated by the methods of the present
invention include CD19-expressing cancers, including, for example,
B-cell lineage malignancies such as, for example, B cell lymphoma
or B cell leukemia, including, but not limited to, non-Hodgkin
lymphoma, chronic lymphocytic leukemia, and acute lymphoblastic
leukemia.
[0011] Also provided are methods for inhibiting the proliferation
or differentiation of tumor cells expressing CD19. Such methods can
include administering to the cells a CD19 binding agent (e.g., an
anti-CD19 full length antibody or antigen-binding fragment thereof
that is not conjugated to a therapeutic agent) or a ligand-drug
conjugate, wherein the ligand unit is a CD19 binding agent (e.g., a
full length antibody or antigen-binding fragment thereof)
conjugated to a cytotoxic, cytostatic and/or therapeutic agent that
specifically binds to and can, for example, inhibit the
proliferation or differentiation of cells expressing human
CD19.
[0012] The present invention encompasses methods for inducing the
depletion of B cells, i.e., peripheral B cells, which are
associated with an immune disorder. Such methods can include
administering to the cells a CD19 binding agent (e.g., an anti-CD19
full length antibody or antigen-binding fragment thereof that is
not conjugated to a therapeutic agent) or a ligand-drug conjugate,
wherein the ligand unit is a CD19 binding agent, (e.g., a full
length antibody or antigen-binding fragment thereof) conjugated to
a cytotoxic, cytostatic and/or therapeutic agent. In some
embodiments, the immune disorder can be rheumatoid arthritis,
systemic lupus erythematosus, multiple sclerosis or inflammatory
bowel disease.
[0013] In another aspect, pharmaceutical compositions are provided
in which the composition comprises a CD19 binding agent (e.g., an
anti-CD19 full length antibody or antigen-binding fragment thereof
that is not conjugated to a therapeutic agent) or a ligand-drug
conjugate, wherein the ligand unit is a CD19 binding agent, (e.g.,
a full length antibody or antigen-binding fragment thereof)
conjugated to a cytotoxic, cytostatic and/or therapeutic agent, and
a pharmaceutically acceptable excipient.
[0014] In another aspect, methods of manufacturing ligand-drug
conjugate compounds are provided. In one aspect, a CD19 binding
agent is conjugated to a cytotoxic, cytostatic and/or therapeutic
agent either directly or through a linker, as described more fully
below.
[0015] The present invention may be more fully understood by
reference to the following detailed description, non-limiting
examples of specific embodiments, and the appended figures and
sequence listing.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1: FACS Results for Binding of CD19 antibodies to
non-human primate spleen cells.
[0017] FIG. 2: Binding Analysis of heavy and light chain variable
regions to CD19.
[0018] FIG. 3: Antitumor activity of naked mBU12 antibody and
mBU12-mcMMAF on Ramos xenograft SCID model. Groups of mice
(5/group) were untreated or received mBU12 naked antibody (1
mg/kg), mBU12 naked antibody (5 mg/kg), mBU12-mcMMAF4 (1 mg/kg) and
mBU12-mcMMAF4 (5 mg/kg) when tumor sizes averaged approximately 100
mm.sup.3. The dose schedule was q4dx3.
[0019] FIG. 4: Antitumor activity of anti-CD19 antibody-drug
conjugates on Ramos tumor model in SCID mice. Groups of mice
(5/group) were untreated or treated with cBU12-mcMMAF8 (3 mg/kg;
complete response 1/5 mice), cHD37-mcMMAF8 (3 mg/kg; complete
response 5/5 mice), c4g7-mcMMAF8 (3 mg/kg; complete response 0/5
mice), or cFMC63-mcMMAF8 (3 mg/kg; complete response 5/5 mice) when
tumor size averaged approximately 100 mm.sup.3. The dose schedule
was q4dx3 iv. In a CR response, the tumor volume is less than 13.5
mm.sup.3 for three consecutive measurements during the course of
the study.
[0020] FIG. 5: Antitumor activity of anti-CD19 antibody-drug
conjugates on Ramos tumor model in SCID mice. Groups of mice
(10/group) were untreated or treated with cBU12-mcMMAF8 (3 mg/kg;
complete response 6/10 mice), hBU12-mcMMAF8 (3 mg/kg; complete
response 10/10 mice; IgG.sub.1 constant region),), hBU12-mcMMAF8 (3
mg/kg; complete response 6/10 mice; IgG.sub.4 constant region),),
cHD37-mcMMAF8 (3 mg/kg; complete response 10/10 mice),
cFMC63-mcMMAF8 (3 mg/kg; complete response 10/10 mice),
c4G7-mcMMAF8 (3 mg/kg; complete response 10/10 mice), or
cAC10-mcMMAF8 (3 mg/kg; complete response 0/10 mice) when tumor
size averaged approximately 100 mm.sup.3. The dose schedule was
q4dx4 iv.
[0021] FIG. 6: Trough levels of cBU12-mcMMAF8 vs cHD37-mcMMAF8.
[0022] FIG. 7: Antitumor activity of anti-CD19 antibody-drug
conjugates in a Ramos tumor model in SCID mice. Groups of mice
(10/group) were untreated or treated with hBU12-mcMMAF8 (3 mg/kg;
complete response 10/10 mice), cHD37-mcMMAF8 (3 mg/kg; complete
response 10/10 mice), or cAC10-mcMMAF8 (3 mg/kg; complete response
0/10 mice) when tumor size averaged approximately 100 mm.sup.3. The
dose schedule was q4dx4 iv.
[0023] FIG. 8: Antitumor activity of anti-CD19 antibody-drug
conjugates in DoHH2 tumor model in SCID mice. Groups of mice
(5/group) were untreated or treated with cAC10-mcMMAF8 (3 mg/kg),
cBU12-mcMMAF8 (3 mg/kg), hBU12-mcMMAF8 (3 mg/kg); cHD37-mcMMAF8 (3
mg/kg) or cFMC63-mcMMAF8 (3 mg/kg) when tumor size averaged
approximately 100 mm.sup.3. The dose schedule was q4dx4, ip.
[0024] FIG. 9: Survival assay for SCID mice treated with anti-CD19
antibody-drug conjugates in Nalm-6 tumor model. Groups of mice
(5/group) were untreated or treated with cAC10-mcMMAF8 (3 mg/kg),
cBU12-mcMMAF8 (3 mg/kg), hBU12-mcMMAF8 (3 mg/kg); cHD37-mcMMAF8 (3
mg/kg) or cFMC63-mcMMAF8 (3 mg/kg). The dose schedule was q4dx4,
iv.
[0025] FIG. 10: Trough levels of hBU12-mcMMAF8 (3 mg/kg) vs
cHD37-mcMMAF8 (3 mg/kg). 5 mice were treated in each group.
[0026] FIG. 11: Antitumor activity of hBU12 antibody-drug
conjugates in Ramos tumor model in SCID mice. Groups of mice
(10/group) were untreated or treated with hBU12-vcMMAE4 (1 mg/kg;
complete response 0/10), hBU12-vcMMAE4 (3 mg/kg; complete response
7/10), hBU12-vcMMAF4 (0.3 mg/kg; complete response 0/10)
hBU12-vcMMAF4 (1 mg/kg; complete response 0/10), hBU12-vcMMAF4 (3
mg/kg; complete response 1/10), hBU12-mcMMAF8 (1 mg/kg; complete
response 0/10), or hBU12-mcMMAF8 (3 mg/kg; complete response
10/10). The dose schedule was q4dx4, iv.
[0027] FIG. 12: Antitumor activity of anti-CD19 antibody-drug
conjugates in DoHH2 tumor model in SCID mice. Groups of mice
(1/group) were treated with varying 1 mg/kg, 3 mg/kg, or 10 mg/kg
of hBU12-vcMMAE4, -mcMMAF4, and -mcMMAF8. The dose schedule was a
single dose, ip.
[0028] FIG. 13: Ramos cells were cultured with anti-CD19 antibodies
cross-linked with a 2-fold excess of goat-anti-mouse ligand drug
conjugate (vcMMAF8). Cultures were incubated for 96 hours and
labeled with 50 .mu.M resazurin. Values are the mean.+-.SD of four
replicates within a single experiment.
[0029] FIG. 14: CD19 and CD21 expression levels and cytotoxicity of
hBU12-vcMMAE4 and hBU12-mcMMAF4 against ALL, CLL, and NHL tumor
cell lines grown in culture.
[0030] FIG. 15: Internalization kinetics and intracellular
trafficking of hBU12-vcMMAE4 on NHL and ALL tumor cell lines.
[0031] FIGS. 16A-16E: Xenograft experiments testing hBU12-vcMMAE4
in models of NHL. SCID mice were implanted subcutaneously with
5.times.10.sup.6 cells of tumor cells in the right flank and
treatment was initiated when the average tumor volume reached 100
mm.sup.3. Treatment was intraperitoneally with 1 or 3 mg/kg, q4dx4
of hBU12-vcMMAE4. There were 7-10 mice per each treatment group.
16A.) Growth curve of the NHL cell line (Burkitt's lymphoma); 16B.)
Growth curve of the follicular lymphoma cell lines DOHH2. 16C.)
Growth curve of the diffuse large B cell lymphoma (DLBCL) cell line
DLCL2. 16D.) Survival curve of mice implanted with the ALL cell
line RS4; 11 via tail vein. Treatment of mice was initiated on day
7 post tumor implantation at a q4dx4, schedule, intraperitoneally.
16E.) Survival curve of mice implanted with the ALL cell line
Nalm-6 via tail vein. Treatment was initiated on day 7 post tumor
implantation, with a single dose of hBU12-vcMMAE4 at the indicated
dose, via intraperitoneal injections.
[0032] FIGS. 17A-17D: Efficacy of hBU12-vcE in rituximab resistant
lymphomas. 17A.) SCID mice were implanted with 5.times.10.sup.6 of
the parental Ramos-P cell lines used to generate rituximab
resistant tumors. Comparable levels of tumor growth inhibiton were
achieved by rituximab (12 mg/kg, q4dx4) and hBU12-vcE (3 mg/kg,
q4dx4) 16B.) Tumor growth curve of rituximab resistant Ramos tumors
(R-Ramos) treated with hBU12-vcE (1 and 3 mg/kg, IP, q4dx4) or
rituximab (12 mg/kg, q4dx4, IP). There was a statistically
significant difference in tumor growth delay induced by these
compounds 17C.) FACS analysis of CD19 and CD20 expression on cells
isolated from Ramos-P (sensitive) and R-Ramos (resistant) tumors.
Comparable expression levels for both antigens were identified in
both tumors. 17D.) Anti-lymphoma effects of hBU12-vcE against
subcutaneously implanted, rituximab resistant Raji2R tumors (NHL,
Burkitt's lymphoma) treated with 1 and 3 mg/kg, q4dx4 of hBU12-vcE
or control conjugate. 9 out of 10 durable regressions were observed
in hBU12-vcE treated mice, while rituximab (12 mg/kg, q4dx4) did
not significantly impact tumor growth. There were 5-10 mice per
group. A durable response (DR) is defined as complete absence of
palpable tumor during the entire experiment.
[0033] FIG. 18: Activity of hBU12 in disseminated model of ALL. The
mice received a single dose of 10 mg/kg hBU12, hBU12-vcE(4) or
hBU12-mcF(4), IP, on day 1 post tumor implanatation. There were 10
mice per group.
[0034] FIG. 19: Limited anti-tumor effects of hBU12 in a
subcutaneous model of NHL (SUDHL4). There were 8-10 mice per
treatment group. The dose schedule was Q4dx4, IP.
DETAILED DESCRIPTION
[0035] The present invention provides, inter alia, CD19 binding
agents that specifically bind to human CD19. The present inventors
have discovered that despite the poor efficacy of ligand-drug
conjugate compounds comprising murine or chimeric BU12 antibodies
conjugated to a cytotoxic agent, effective ligand-drug conjugate
compounds that target human CD19 can be developed. Specifically,
the present inventors have designed ligand-drug conjugate compounds
comprising humanized BU12 antibodies as the ligand unit conjugated
to a cytotoxic agent. These ligand-drug conjugate compounds have
surprising efficacy given their murine and chimeric
counterparts.
[0036] In certain aspects, the CD19 binding agents of the present
invention comprise at least one of the CDR regions of the antibody
mBU12. In certain aspects, the CD19 binding agents comprise all six
of the CDR regions of the mBU12 antibody. In some embodiments, the
CDR regions have at least one, at least two, or at least three
conservative amino acid substitutions of a CDR of antibody
mBU12.
[0037] In certain aspects, the CD19 binding agents of the present
invention comprise an antibody heavy chain variable region and/or
an antibody light chain variable region, including derivatives
thereof.
[0038] In some aspects, the compositions and methods relate to
antibodies, including antibody derivatives, that bind to CD19. In
certain aspects, the anti-CD19 antibodies and derivatives comprise
the amino acid sequence of a humanized heavy chain variable region
and/or a humanized light chain variable region of antibody BU12,
including derivatives thereof. In certain aspects, the anti-CD19
antibodies and derivatives comprise at least one, at least two, at
least three, at least four, at least five, or all six of the CDR
regions of antibody mBU12. In some embodiments, the anti-CD19
antibodies include at least one immunoglobulin constant region
domain, or an entire constant region of an antibody, such as a
human constant region or, optionally, a functionally active portion
thereof. In some embodiments, the antibody constant region or
domain(s) is of the IgG class. In some embodiments, the antibody
constant domain is IgG1, IgG2, or IgG1V1.
[0039] In certain aspects, the compositions and methods relate to
antibodies, including antibody derivatives, that bind to CD19 and
are conjugated to cytotoxic, cytostatic and/or therapeutic agents.
In certain embodiments, the antibodies have altered glycosylation
patterns.
[0040] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections which follow.
Definitions and Abbreviations
[0041] When a trade name is used herein, reference to the trade
name also refers to the product formulation, the generic drug, and
the active pharmaceutical ingredient(s) of the trade name product,
unless otherwise indicated by context.
[0042] The terms "CD19 binding agent" and "anti-CD19 binding agent"
as used herein refers to a molecule that specifically binds to
CD19. Examples can include a full length anti-CD19 antibody, a
fragment of a full length anti-CD19 antibody, or other agent that
includes an antibody heavy and/or light chain variable region, and
derivatives thereof.
[0043] The terms "specific binding" and "specifically binds" mean
that the CD19 binding agent will react, in a highly selective
manner, with its corresponding target, CD19 and not with the
multitude of other antigens. Typically, the C19 binding agent binds
with an affinity of at least about 1.times.10.sup.-7 10.sup.-1, and
preferably 10.sup.-8 M to 10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M,
or 10.sup.-12 M and binds to the predetermined antigen with an
affinity that is at least two-fold greater than its affinity for
binding to a non-specific antigen (e.g., BSA, casein) other than
the predetermined antigen or a closely-related antigen
[0044] As used herein, the term "functional," in the context of a
CD19 binding agent, indicates that the binding agent is capable of
specifically binding to CD19.
[0045] The terms "inhibit" or "inhibition of" as used herein means
to reduce by a measurable amount, or to prevent entirely.
[0046] The term "deplete," in the context of the effect of a CD19
binding agent on CD19-expressing cells, refers to a reduction in
the number of or elimination of the CD19-expressing cells.
[0047] "Native antibodies" and "native immunoglobulins" are defined
herein as heterotetrameric glycoproteins, typically of about
150,000 daltons, composed of two light (L) chain and two heavy (H)
chains. Each light chain is covalently linked to a heavy chain by a
disulfide bond to form a heterodimer. The heterotetramer is formed
by covalent disulfide linkage between the two heavy chains of such
heterodimers. Although the light and heavy chains are linked
together by a disulfide bond, the number of disulfide linkages
between the two heavy chains varies by immunoglobulin (Ig) isotype.
Each heavy and light chain also has regularly spaced intrachain
disulfide bridges. Each heavy chain has at the amino-terminus a
variable domain (V.sub.H), followed by three or four constant
domains (C.sub.H1, C.sub.H2, C.sub.H3, and/or C.sub.H4, as
appropriate for the antibody type), as well as a hinge (J) region
between C.sub.H1 and C.sub.H2. Each light chain has two domains, an
amino-terminal variable domain (V.sub.L) and a carboxy-terminal
constant domain (C.sub.L). The V.sub.L domain associates
non-covalently with the V.sub.H domain, whereas the C.sub.L domain
is commonly covalently linked to the C.sub.H1 domain via a
disulfide bond. Particular amino acid residues are believed to form
an interface between the light and heavy chain variable domains
(see, e.g., Chothia et al., 1985, J. Mol. Biol. 186:651-663).
[0048] The term "hypervariable" refers to certain sequences within
the variable domains of an immunoglobulin that differ extensively
in sequence among antibodies and contain residues that are directly
involved in the binding and specificity of each particular antibody
for its specific antigenic determinant. Hypervariability, both in
the light chain and the heavy chain variable domains, is
concentrated in three segments known as complementarity determining
regions (CDRs) or hypervariable loops (HVLs). The locations of the
CDRs are defined by sequence comparison in Kabat et al., 1991, In:
Sequences of Proteins of Immunological Interest, 5.sup.th Ed.
Public Health Service, National Institutes of Health, Bethesda,
Md., whereas HVLs are structurally defined according to the
three-dimensional structure of the variable domain, as described by
Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917. As defined by
Kabat, CDR-L1 is positioned at about residues 24-34, CDR-L2 at
about residues 50-56, and CDR-L3 at about residues 89-97 in the
light chain variable domain. CDR-H1 is positioned at about residues
31-35, CDR-H2 at about residues 50-65, and CDR-H3 at about 95-102
in the heavy chain variable domain.
[0049] The three CDRs within each of the heavy and light chains are
separated by framework regions (FR), which contain sequences that
tend to be less variable. From the amino terminus to the carboxy
terminus of the heavy and light chain variable domains, the FRs and
CDRs are arranged in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3,
and FR4. The largely .beta.-sheet configuration of the FRs brings
the CDRs within each of the chains to close proximity to each other
as well as to the CDRs from the other chain. The resulting
conformation contributes to the antigen binding site (see, e.g.,
Kabat et al., 1991, NIH Publ. No. 91-3242, Vol. I, pages 647-669),
although not all CDR residues are necessarily directly involved in
antigen binding.
[0050] FR residues and Ig constant domains are typically not
directly involved in antigen binding, but may contribute to antigen
binding or mediate antibody effector function. Some FR residues can
have a significant effect on antigen binding in at least three
ways: by noncovalently binding directly to an epitope, by
interacting with one or more CDR residues, and by affecting the
interface between the heavy and light chains. In some embodiments,
the constant domains mediate various Ig effector functions, such as
participation of the antibody in antibody dependent cellular
cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and/or
antibody dependent cellular phagocytosis (ADCP).
[0051] The light chains of vertebrate immunoglobulins are assigned
to one of two clearly distinct classes, kappa (.kappa.) and lambda
(.lamda.), based on the amino acid sequence of the constant domain.
By comparison, the heavy chains of mammalian immunoglobulins are
assigned to one of five major classes, according to the sequence of
the constant domains: IgA, IgD, IgE, IgG, and IgM. IgG and IgA are
further divided into subclasses (isotypes), e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, and IgG.sub.4; and IgA.sub.1, and IgA.sub.2,
respectively. The heavy chain constant domains that correspond to
the different classes of immunoglobulins are called .alpha.,
.delta., .epsilon., .gamma., and .mu., respectively. The subunit
structures and three-dimensional configurations of the classes of
native immunoglobulins are well known.
[0052] The terms "antibody", "anti-CD19 antibody", "humanized
anti-CD19 antibody", and "variant humanized anti-CD19 antibody" are
used herein in the broadest sense and specifically encompass
full-length and native antibodies, monoclonal antibodies (including
full-length monoclonal antibodies), polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and
antibody fragments thereof, such as variable domains and other
portions of antibodies that exhibit a desired biological activity
(e.g., CD19 binding). The terms "anti-CD19 antibody fragment",
"humanized anti-CD19 antibody fragment", and "variant humanized
anti-CD19 antibody fragment" refer to a portion of a full-length
anti-CD19 antibody in which a variable region or a functional
capability is retained, for example, specific CD19 epitope binding.
Examples of antibody fragments include, but are not limited to, a
Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragment, a diabody, a
linear antibody, a minibody and a multispecific antibody formed
from antigen-binding antibody fragments. Antibody fragments are
specifically included within the definition of "antibody".
[0053] The terms "monoclonal antibody" or "mAb" refer to an
antibody obtained from a population of substantially homogeneous
antibodies; that is, the individual antibodies comprising the
population are identical except for naturally occurring mutations
that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic
determinant, also referred to as an epitope. The modifier
"monoclonal" is indicative of a substantially homogeneous
population of antibodies directed to the identical epitope and is
not to be construed as requiring production of the antibody by any
particular method. Monoclonal antibodies can be made by any
technique or methodology known in the art; for example, the
hybridoma method first described by Kohler et al., 1975, Nature
256:495, or recombinant DNA methods known in the art (see, e.g.,
U.S. Pat. No. 4,816,567). In another example, monoclonal antibodies
also can be isolated from phage antibody libraries, using
techniques described in Clackson et al., 1991, Nature 352: 624-628,
and Marks et al., 1991, J. Mol. Biol. 222: 581-97.
[0054] The term "chimeric" antibody, as used herein, refers to a
type of monoclonal antibody in which a portion of or the complete
amino acid sequence in one or more regions or domains of the heavy
and/or light chain is identical with, homologous to, or a variant
of, the corresponding sequence in a monoclonal antibody from
another species or belonging to another immunoglobulin class or
isotype, or from a consensus sequence. An example of a chimeric
antibody is one which has a variable region derived from a
non-human monoclonal antibody and a human IgG immunoglobulin
constant region. Chimeric antibodies include fragments of such
antibodies, provided that the antibody fragment exhibits the
desired biological activity of its parent antibody, for example
binding to the same epitope (see, e.g., U.S. Pat. No. 4,816,567;
and Morrison et al., 1984, Proc. Natl. Acad. Sci. USA
81:6851-6855). Methods for producing chimeric antibodies are known
in the art. (See, e.g., Morrison, 1985, Science 229:1202; Oi et
al., 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol.
Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and
4,816,397.)
[0055] A "single-chain Fv" or "scFv" antibody fragment is a single
chain Fv variant comprising the V.sub.H and V.sub.L domains of an
antibody in which the domains are present in a single polypeptide
chain and which is capable of recognizing and binding antigen. The
scFv polypeptide optionally contains a polypeptide linker
positioned between the V.sub.H and V.sub.L domains that enables the
scFv to form a desired three-dimensional structure for antigen
binding, (see, e.g., PlUckthun, 1994, In The Pharmacology of
Monoclonal Antibodies, Vol. 113, Rosenburg and Moore eds.,
Springer-Verlag, New York, pp. 269-315).
[0056] The term "diabody" refers to a small antibody fragment
having two antigen-binding sites. Each fragment contains a heavy
chain variable domain (V.sub.H) concatenated to a light chain
variable domain (V.sub.L) to form a V.sub.H-V.sub.L or
V.sub.L-V.sub.H polypeptide. By using a linker that is too short to
allow pairing between the two domains on the same chain, the linked
V.sub.H-V.sub.L domains are forced to pair with complementary
domains of another chain, creating two antigen-binding sites.
Diabodies are described more fully, for example, in EP 0 404 097;
WO 93/11161; and Hollinger et al., 1993, Proc. Natl. Acad. Sci. USA
90:6444-6448.
[0057] The term "linear antibody" refers to an antibody that has a
pair of tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) that
form a pair of antigen binding regions. Linear antibodies can be
bispecific or monospecific, as described in Zapata et al., 1995,
Protein Eng. 8(10):1057-1062.
[0058] A "humanized" antibody for the purposes herein is an
immunoglobulin amino acid sequence variant or fragment thereof
which is capable of binding to a predetermined antigen and which
comprises a framework region having substantially the amino acid
sequence of a human immunoglobulin and a CDR having substantially
the amino acid sequence of a non-human immunoglobulin.
[0059] Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are referred to herein as "import"
residues, which are typically taken from an "import" antibody
domain, particularly a variable domain. An import residue,
sequence, or antibody has a desired affinity and/or specificity, or
other desirable antibody biological activity as discussed
herein.
[0060] In general, the humanized antibody will comprise
substantially all of at least one, and sometimes two, variable
domains in which all or substantially all of the CDR regions
correspond to those of a non-human immunoglobulin and all or
substantially all of the FR regions are those of a human
immunoglobulin sequence from, e.g., a consensus or germline
sequence. The humanized antibody optionally also will comprise at
least a portion of an immunoglobulin Fc region, typically that of a
human immunoglobulin. In certain aspects, the antibody will contain
both the light chain variable region as well as the heavy chain
variable region. The antibody also may include the C.sub.H1, hinge
(J), C.sub.H2, C.sub.H3, and/or C.sub.H4 regions of the heavy
chain, and the C.sub.L region of the light chain, as
appropriate.
[0061] The humanized antibody will be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including IgG.sub.1, IgG.sub.2, IgG.sub.3 and IgG.sub.4,
and IgA.sub.1, and IgA.sub.2. The choice of which immunoglobulin
class or isotype will depend, in part, on the desired effector
function. For example, the ability of human immunoglobulins to
mediate CDC and ADCC/ADCP is generally in the order of
IgM.apprxeq.IgG.sub.1.apprxeq.IgG.sub.3>IgG.sub.2>IgG.sub.4
and IgG.sub.1.apprxeq.IgG.sub.3>IgG.sub.2/IgM/IgG.sub.4,
respectively. The humanized antibody may comprise sequences from
more than one class or isotype, and selecting particular constant
domains to optimize desired effector functions is within the
ordinary skill in the art. The humanized antibody may or may not
have effector function.
[0062] The FRs and CDRs of the humanized antibody need not
correspond precisely to the parental sequences, e.g., the import
CDR or the consensus FR may be altered by substitution, insertion
or deletion of at least one residue so that the CDR or FR residue
at that site does not correspond to either the consensus or the
import antibody. Typically, such changes will not be extensive.
Usually, at least 75% of the humanized antibody residues will
correspond to those of the parental FR and CDR sequences, more
often 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least
99%.
[0063] A "therapeutic agent" is an agent that exerts a cytotoxic,
cytostatic, and/or immunomodulatory effect on cancer cells or
activated immune cells. Examples of therapeutic agents include
cytotoxic agents, chemotherapeutic agents, cytostatic agents, and
immunomodulatory agents. Illustrative therapeutic agents include
chemotherapeutic drugs, cytotoxins, immunomodulators, chelators,
boron compounds, photoactive agents, photoactive dyes, steroids,
radioisotopes and the like.
[0064] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer.
[0065] A "cytotoxic effect" refers to the depletion, elimination
and/or the killing of a target cell(s). A "cytotoxic agent" refers
to an agent that has a cytotoxic and/or cytostatic effect on a
cell. The term is intended to include radioactive isotopes (e.g.,
I.sup.131, I.sup.125, Y.sup.90, and Re.sup.186), chemotherapeutic
agents, and toxins such as enzymatically active toxins of
bacterial, fungal, plant, or animal origin, and fragments
thereof.
[0066] A "cytostatic effect" refers to the inhibition of cell
proliferation. A "cytostatic agent" refers to an agent that has a
cytostatic effect on a cell, thereby inhibiting the growth and/or
expansion of a specific subset of cells.
[0067] The term "label" refers to a detectable compound or
composition that is conjugated directly or indirectly to a binding
agent (e.g., an antibody). The label may itself be detectable
(e.g., a radioisotope label or a fluorescent label) or, in the case
of an enzymatic label, may catalyze a chemical alteration of a
substrate compound or composition that is detectable. Labeled CD19
binding agents can be prepared and used in various applications
including in vitro and in vivo diagnostics. Useful labels include
diagnostic agents such as contrast agents (such as for magnetic
resonance imaging, computed tomography or ultrasound, e.g.,
manganese, iron or gadolinium).
[0068] An "isolated" nucleic acid molecule is a nucleic acid
molecule that is identified and separated from at least one
contaminant nucleic acid molecule with which it is ordinarily
associated in the natural source of the nucleic acid. An isolated
nucleic acid molecule is other than in the form or setting in which
it is found in nature. Isolated nucleic acid molecules therefore
are distinguished from the nucleic acid molecule as it exists in
natural cells. However, an isolated nucleic acid molecule includes
a nucleic acid molecule contained in cells that ordinarily express
the antibody where, for example, the nucleic acid molecule is in a
chromosomal location different from that of natural cells.
[0069] The term "control sequence" refers to a polynucleotide
sequence necessary for expression of an operably linked coding
sequence in a particular host organism. The control sequences
suitable for use in prokaryotic cells include, for example, a
promoter, operator and ribosome binding site sequences. Eukaryotic
control sequences include, but are not limited to, promoters,
polyadenylation signals, and enhancers. These control sequences can
be utilized for expression and production of CD19 binding agents in
prokaryotic and eukaryotic host cells.
[0070] A nucleic acid sequence is "operably linked" when it is
placed into a functional relationship with another nucleic acid
sequence. For example, a nucleic acid presequence or secretory
leader is operably linked to a nucleic acid encoding a polypeptide
if it is expressed as a preprotein that participates in the
secretion of the polypeptide; a promoter or enhancer is operably
linked to a coding sequence if it affects the transcription of the
sequence; or a ribosome binding site is operably linked to a coding
sequence if it is positioned so as to facilitate translation.
Generally, "operably linked" means that the nucleic acid sequences
being linked are contiguous, and, in the case of a secretory
leader, contiguous and in reading frame. However, enhancers are
optionally contiguous. Linking can be accomplished, for example, by
ligation at convenient restriction sites. If such sites do not
exist, synthetic oligonucleotide adaptors, linkers or other methods
known in the art can be used.
[0071] The term "polypeptide" refers to a polymer of amino acids
and its equivalent and does not refer to a specific length of a
product; thus, "peptides" and "proteins" are included within the
definition of a polypeptide. Also included within the definition of
polypeptides are "antibodies" as defined herein. A "polypeptide
region" refers to a segment of a polypeptide, which segment may
contain, for example, one or more domains or motifs (e.g., a
polypeptide region of an antibody can contain, for example, one or
more complementarity determining regions (CDRs)). The term
"fragment" refers to a portion of a polypeptide typically having at
least 20 contiguous or at least 50 contiguous amino acids of the
polypeptide.
[0072] Unless otherwise indicated by context, a "derivative" is a
polypeptide or fragment thereof having one or more non-conservative
or conservative amino acid substitutions relative to a second
polypeptide (also referred to as a "variant"); or a polypeptide or
fragment thereof that is modified by covalent attachment of a
second molecule such as, e.g., by attachment of a heterologous
polypeptide, or by glycosylation, acetylation, phosphorylation, and
the like. Further included within the definition of "derivative"
are, for example, polypeptides containing one or more analogs of an
amino acid (e.g., unnatural amino acids and the like), polypeptides
with unsubstituted linkages, as well as other modifications known
in the art, both naturally and non-naturally occurring.
[0073] An "isolated" polypeptide is one which has been identified
and separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the polypeptide, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. An isolated polypeptide
includes an isolated antibody, or a fragment or derivative
thereof.
[0074] In certain embodiments, the polypeptide will be purified (1)
to greater than 95% by weight of polypeptide as determined by the
Lowry method, and in other aspects to more than 99% by weight, (2)
to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of a spinning cup
sequenator, or (3) to homogeneity by SDS-PAGE under reducing or
nonreducing conditions using Coomassie blue or, preferably, silver
stain. "Isolated antibody" includes the antibody in situ within
recombinant cells since at least one component of the antibody's
natural environment will not be present.
[0075] The term "heterologous," in the context of a polypeptide,
means from a different source (e.g., a cell, tissue, organism, or
species) as compared with another polypeptide, so that the two
polypeptides are different. Typically, a heterologous polypeptide
is from a different species.
[0076] In the context of immunoglobulin polypeptides, or fragments
thereof, as defined above, "conservative substitution" means one or
more amino acid substiutions that do not substantially reduce
specific binding (e.g., as measured by the K.sub.D) of the
immunoglobulin polypeptide or fragment thereof to an antigen (e.g.,
substitutions that increase binding, that do not significantly
alter binding, or that reduce binding by no more than about 40%,
typically no more than about 30%, more typically no more than about
20%, even more typically no more than about 10%, or most typically
no more than about 5%, as determined by standard binding assays
such as, e.g., ELISA).
[0077] The terms "identical" or "percent identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of nucleotides or amino acid residues that are
the same, when compared and aligned for maximum correspondence. To
determine the percent identity, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions (e.g., overlapping
positions).times.100). In some embodiments, the two sequences are
the same length.
[0078] The term "substantially identical," in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%,
at least 98% identity, or at least 99% identity (e.g., as
determined using one of the methods set forth infra).
[0079] The terms "similarity" or "percent similarity" in the
context of two or more polypeptide sequences, refer to two or more
sequences or subsequences that have a specified percentage of amino
acid residues that are the same or conservatively substituted when
compared and aligned for maximum correspondence, as measured using
one of the methods set forth infra. By way of example, a first
amino acid sequence can be considered similar to a second amino
acid sequence when the first amino acid sequence is at least 50%,
60%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% identical, or conservatively substituted, to the second amino
acid sequence when compared to an equal number of amino acids as
the number contained in the first sequence, or when compared to an
alignment of polypeptides that has been aligned by, for example,
one of the methods set forth infra.
[0080] In the context of CD19 binding agents of the present
invention, a protein that has one or more polypeptide regions
substantially identical or substantially similar to one or more
antigen-binding regions (e.g., a heavy or light chain variable
region, or a heavy or light chain CDR) of an anti-CD19 antibody
retains specific binding to an epitope of CD19 recognized by the
anti-CD19 antibody, as determined using any of various standard
immunoassays known in the art or as referred to herein.
[0081] The determination of percent identity or percent similarity
between two sequences can be accomplished using a mathematical
algorithm. A non-limiting example of a mathematical algorithm
utilized for the comparison of two sequences is the algorithm of
Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268,
modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci.
USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST
and XBLAST programs of Altschul et al., 1990, J. Mol. Biol.
215:403-410. BLAST nucleotide searches can be performed with the
NBLAST program, score=100, wordlength=12, to obtain nucleotide
sequences homologous to a nucleic acid encoding a protein of
interest. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3, to obtain amino acid sequences
homologous to a protein of interest. To obtain gapped alignments
for comparison purposes, Gapped BLAST can be utilized as described
in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.
Alternatively, PSI-Blast can be used to perform an iterated search
which detects distant relationships between molecules (id.). When
utilizing BLAST, Gapped BLAST, and PSI-BLAST programs, the default
parameters of the respective programs (e.g., XBLAST and NBLAST) can
be used. Another non-limiting example of a mathematical algorithm
utilized for the comparison of sequences is the algorithm of Myers
and Miller, CABIOS (1989). Such an algorithm is incorporated into
the ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 can be used.
Additional algorithms for sequence analysis are known in the art
and include ADVANCE and ADAM as described in Torellis and Robotti,
1994, Comput. Appl. Biosci. 10:3-5; and FASTA described in Pearson
and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444-8. Within
FASTA, ktup is a control option that sets the sensitivity and speed
of the search. If ktup=2, similar regions in the two sequences
being compared are found by looking at pairs of aligned residues;
if ktup=1, single aligned amino acids are examined. ktup can be set
to 2 or 1 for protein sequences, or from 1 to 6 for DNA sequences.
The default if ktup is not specified is 2 for proteins and 6 for
DNA. Alternatively, protein sequence alignment may be carried out
using the CLUSTAL W algorithm, as described by Higgins et al.,
1996, Methods Enzymol. 266:383-402.
[0082] Optionally, any two antibody sequences can be aligned, for
example to determine percent identity, by using the Kabat numbering
system so that each amino acid in one antibody sequence is aligned
with the amino acid in the other sequence that has the same Kabat
number. After alignment, if a subject antibody region (e.g., the
entire mature variable region of a heavy or light chain) is being
compared with the same region of a reference antibody, the
percentage sequence identity between the subject and reference
antibody regions is the number of positions occupied by the same
amino acid in both the subject and reference antibody region
divided by the total number of aligned positions of the two
regions, with gaps not counted, multiplied by 100 to convert to
percentage.
[0083] "Effector cell" as used herein refers to a cell that
expresses a surface receptor for the Fc region of an immunoglobulin
(FcR). For example, cells that express surface FcR for IgGs
including Fc.gamma.RIII (CD16), Fc.gamma.RII (CD32) and Fc.gamma.RI
(CD64) can act as effector cells. Such effector cells include
monocytes, macrophages, natural killer (NK) cells, neutrophils and
eosinophils.
[0084] The term "antibody effector function(s)" as used herein
refers to a function contributed by an Fc region(s) of an Ig. Such
function can be effected by, for example, binding of an Fc effector
region (s) to an Fc receptor on an immune cell with phagocytic or
lytic activity or by binding of an Fc effector region(s) to
components of the complement system. The CD19 binding agents of the
present invention may or may not have effector function.
[0085] A "disorder", as used herein, and the terms "CD19-associated
disorder" and "CD19-associated disease" refer to any condition that
would benefit from treatment with a CD19 binding agent described
herein. This includes chronic and acute disorders or diseases
including those pathological conditions that predispose the mammal
to the disorder in question. Non-limiting examples or disorders to
be treated herein include CD19 expressing cancers, including
hematological malignancies, benign and malignant tumors, leukemias
and lymphoid malignancies, as well as inflammatory, angiogenic and
immunologic disorders. Specific examples of disorders are disclosed
infra
[0086] The terms "treatment" and "therapy", and the like, as used
herein, are meant to include therapeutic or suppressive measures
for a disease or disorder leading to any clinically desirable or
beneficial effect, including, but not limited to, alleviation or
relief of one or more symptoms, regression, slowing or cessation of
progression of the disease or disorder associated with CD19
expression, such as a cancer. For example, treatment can include a
decrease or elimination of a clinical or diagnostic symptom of a
CD19-expressing disorder after the onset of the clinical or
diagnostic symptom by administration of an anti-CD19 antibody or
other CD19 binding agent to a subject. Treatment can be evidenced
as a decrease in the severity of a symptom, the number of symptoms,
or frequency of relapse.
[0087] Except when noted, the terms "subject" or "patient" are used
interchangeably and refer to mammals such as human patients and
non-human primates, as well as experimental animals such as
rabbits, dogs, cats, rats, mice, and other animals. Accordingly,
the term "subject" or "patient" as used herein means any mammalian
patient or subject to which the CD19 binding agents of the
invention can be administered. Subjects of the present invention
include those that have been diagnosed with a CD19 expressing
cancer, including, for example, B cell lymphoma or B cell leukemia,
including, but not limited to, non-Hodgkin lymphoma, chronic
lymphocytic leukemia, and acute lymphoblastic leukemia. In certain
embodiments, the subject will have a refractory or relapsed CD19
expressing cancer
[0088] A subject with a refractory CD19 expressing cancer is a
subject who does not respond to therapy, i.e., the subject
continues to experience disease progresssion despite therapy.
[0089] A subject with a relapsed CD19 expressing cancer is a
subject who has responded to the therapy at one point, but has had
a reoccurence or further progression of disease following the
response.
[0090] The term "effective amount" refers to the amount of a CD19
binding agent or ligand-drug conjugate that is sufficient to
inhibit the occurrence or ameliorate one or more clinical or
diagnostic symptoms of a CD19-associated disorder in a subject. An
effective amount of an agent is administered according to the
methods described herein in an "effective regimen." The term
"effective regimen" refers to a combination of amount of the agent
and dosage frequency adequate to accomplish treatment or prevention
of a CD19-associated disorder.
[0091] The term "pharmaceutically acceptable" as used herein refers
to those compounds, materials, compositions, and/or dosage forms
that are, within the scope of sound medical judgment, suitable for
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other
problems or complications commensurate with a reasonable
benefit/risk ratio. The term "pharmaceutically compatible
ingredient" refers to a pharmaceutically acceptable diluent,
adjuvant, excipient, or vehicle with which a CD19 binding agent or
a ligand-drug conjugate compound is administered.
[0092] The term "pharmaceutically compatible ingredient" refers to
a pharmaceutically acceptable diluent, adjuvant, excipient, or
vehicle with which a CD19 binding agent is administered.
[0093] The term "compound" refers to and encompasses the chemical
compound itself as well as, whether explicitly stated or not, and
unless the context makes clear that the following are to be
excluded: amorphous and crystalline forms of the compound,
including polymorphic forms, where these forms may be part of a
mixture or in isolation; free acid and free base forms of the
compound, which are typically the forms shown in the structures
provided herein; isomers of the compound, which refers to optical
isomers, and tautomeric isomers, where optical isomers include
enantiomers and diastereomers, chiral isomers and non-chiral
isomers, and the optical isomers include isolated optical isomers
as well as mixtures of optical isomers including racemic and
non-racemic mixtures; where an isomer may be in isolated form or in
a mixture with one or more other isomers; isotopes of the compound,
including deuterium- and tritium-containing compounds, and
including compounds containing radioisotopes, including
therapeutically- and diagnostically-effective radioisotopes;
multimeric forms of the compound, including dimeric, trimeric, etc.
forms; salts of the compound, preferably pharmaceutically
acceptable salts, including acid addition salts and base addition
salts, including salts having organic counterions and inorganic
counterions, and including zwitterionic forms, where if a compound
is associated with two or more counterions, the two or more
counterions may be the same or different; and solvates of the
compound, including hemisolvates, monosolvates, disolvates, etc.,
including organic solvates and inorganic solvates, said inorganic
solvates including hydrates; where if a compound is associated with
two or more solvent molecules, the two or more solvent molecules
may be the same or different. In some instances, reference made
herein to a compound of the invention will include an explicit
reference to one or of the above forms, e.g., salts and/or
solvates, however, this reference is for emphasis only, and is not
to be construed as excluding other of the above forms as identified
above.
[0094] As used herein, "pharmaceutically acceptable salts" refer to
derivatives of the disclosed compounds wherein the parent compound
is modified by making acid or base salts thereof. Examples of
pharmaceutically acceptable salts include, but are not limited to,
mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic
acids; and the like. The pharmaceutically acceptable salts include
the conventional non-toxic salts or the quaternary ammonium salts
of the parent compound formed, for example, from non-toxic
inorganic or organic acids. For example, such conventional
non-toxic salts include those derived from inorganic acids such as
hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric
and the like; and the salts prepared from organic acids such as
acetic, propionic, succinic, glycolic, stearic, lactic, malic,
tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,
phenylacetic, glutamic, benzoic, salicylic, sulfanilic,
2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane
disulfonic, oxalic, isethionic, and the like. These physiologically
acceptable salts are prepared by methods known in the art, e.g., by
dissolving the free amine bases with an excess of the acid in
aqueous alcohol, or neutralizing a free carboxylic acid with an
alkali metal base such as a hydroxide, or with an amine
[0095] Unless otherwise noted, the term "alkyl" refers to a
saturated straight or branched hydrocarbon having from about 1 to
about 20 carbon atoms (and all combinations and subcombinations of
ranges and specific numbers of carbon atoms therein), with from
about 1 to about 8 carbon atoms being preferred. Examples of alkyl
groups are methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl,
sec-butyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl,
2-methyl-2-butyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl,
3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl,
2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl,
4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl,
2,3-dimethyl-2-butyl, and 3,3-dimethyl-2-butyl.
[0096] Alkyl groups, whether alone or as part of another group, can
be optionally substituted with one or more groups, preferably 1 to
3 groups (and any additional substituents selected from halogen),
including, but not limited to, halogen, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl, and
wherein said optionally substituted O--(C.sub.1-C.sub.8 alkyl),
optionally substituted --O--(C.sub.2-C.sub.8 alkenyl), optionally
substituted --O--(C.sub.2-C.sub.8 alkynyl), optionally substituted
aryl, optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, and optionally substituted
--C.sub.2-C.sub.8 alkynyl groups can be optionally further
substituted with one or more groups including, but not limited to,
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl.
[0097] Unless otherwise noted, the terms "alkenyl" and "alkynyl"
refer to straight and branched carbon chains having from about 2 to
about 20 carbon atoms (and all combinations and subcombinations of
ranges and specific numbers of carbon atoms therein), with from
about 2 to about 8 carbon atoms being preferred. An alkenyl chain
has at least one double bond in the chain and an alkynyl chain has
at least one triple bond in the chain. Examples of alkenyl groups
include, but are not limited to, ethylene or vinyl, allyl,
-1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,
-3-methyl-1-butenyl, -2-methyl-2-butenyl, and
-2,3-dimethyl-2-butenyl. Examples of alkynyl groups include, but
are not limited to, acetylenic, propargyl, acetylenyl, propynyl,
-1-butynyl, -2-butynyl, -1-pentynyl, -2-pentynyl, and -3-methyl-1
butynyl.
[0098] Alkenyl and alkynyl groups, whether alone or as part of
another group, can be optionally substituted with one or more
groups, preferably 1 to 3 groups (and any additional substituents
selected from halogen), including but not limited halogen,
optionally substituted --O--(C.sub.1-C.sub.8 alkyl), optionally
substituted --O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkyenl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl and
wherein said optionally substituted --O--(C.sub.1-C.sub.8 alkyl),
optionally substituted --O--(C.sub.2-C.sub.8 alkenyl), optionally
substituted --O--(C.sub.2-C.sub.8 alkynyl), optionally substituted
aryl, optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, and optionally substituted
--C.sub.2-C.sub.8 alkynyl groups can be optionally further
substituted with one or more substituents including, but not
limited to, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl.
[0099] Unless otherwise noted, the term "alkylene" refers to a
saturated branched or straight chain hydrocarbon radical having
from about 1 to about 20 carbon atoms (and all combinations and
subcombinations of ranges and specific numbers of carbon atoms
therein), with from about 1 to about 8 carbon atoms being preferred
and having two monovalent radical centers derived by the removal of
two hydrogen atoms from the same or two different carbon atoms of a
parent alkane. Typical alkylenes include, but are not limited to,
methylene, ethylene, propylene, butylene, pentylene, hexylene,
heptylene, ocytylene, nonylene, decalene, 1,4-cyclohexylene, and
the like. Alkylene groups, whether alone or as part of another
group, can be optionally substituted with one or more groups,
preferably 1 to 3 groups (and any additional substituents selected
from halogen), including, but not limited to, halogen, optionally
substituted --O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl and
wherein said optionally substituted O--(C.sub.1-C.sub.8 alkyl),
optionally substituted --O--(C.sub.2-C.sub.8 alkenyl), optionally
substituted --O--(C.sub.2-C.sub.8 alkynyl), optionally substituted
aryl, optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, and optionally substituted
--C.sub.2-C.sub.8 alkynyl groups can be further optionally
substituted with one or more substituents including, but not
limited to, C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl.
[0100] Unless otherwise noted, the term "alkenylene" refers to an
optionally substituted alkylene group containing at least one
carbon-carbon double bond. Exemplary alkenylene groups include, for
example, ethenylene (--C.sub.H.dbd.C.sub.H--) and propenylene
(--C.sub.H.dbd.CHCH.sub.2--).
[0101] Unless otherwise noted, the term "alkynylene" refers to an
optionally substituted alkylene group containing at least one
carbon-carbon triple bond. Exemplary alkynylene groups include, for
example, acetylene (--C.ident.C--), propargyl
(--CH.sub.2C.ident.C--), and 4-pentynyl
(--CH.sub.2CH.sub.2CH.sub.2C.ident.CH--).
[0102] Unless otherwise noted, the term "aryl" refers to a
monovalent aromatic hydrocarbon radical of 6-20 carbon atoms (and
all combinations and subcombinations of ranges and specific numbers
of carbon atoms therein) derived by the removal of one hydrogen
atom from a single carbon atom of a parent aromatic ring system.
Some aryl groups are represented in the exemplary structures as
"Ar". Typical aryl groups include, but are not limited to, radicals
derived from benzene, substituted benzene, phenyl, naphthalene,
anthracene, biphenyl, and the like.
[0103] An aryl group, whether alone or as part of another group,
can be optionally substituted with one or more, preferably 1 to 5,
or even 1 to 2 groups including, but not limited to, halogen,
optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, --NO.sub.2, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl and
wherein said optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), and optionally substituted aryl
groups can be further optionally substituted with one or more
substituents including, but not limited to, C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, halogen,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'', --OC(O)R'',
--C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'', --C(O)N(R'').sub.2,
--NHC(O)R'', --SR'', --SO.sub.3R'', --S(O).sub.2R'', --S(O)R'',
--OH, --N.sub.3, --NH.sub.2, --NH(R''), --N(R'').sub.2 and --CN,
where each R'' is independently selected from H, --C.sub.1-C.sub.8
alkyl, --C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, or
aryl.
[0104] Unless otherwise noted, the term "arylene" refers to an
optionally substituted aryl group which is divalent (i.e., derived
by the removal of two hydrogen atoms from the same or two different
carbon atoms of a parent aromatic ring system) and can be in the
ortho, meta, or para configurations as shown in the following
structures with phenyl as the exemplary aryl group:
##STR00001##
Typical "--(C.sub.1-C.sub.8 alkylene)aryl," "--(C.sub.2-C.sub.8
alkenylene)aryl, "and --(C.sub.2-C.sub.8 alkynylene)aryl groups
include, but are not limited to, benzyl, 2-phenylethan-1-yl,
2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,
2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and
the like.
[0105] Unless otherwise noted, the term "heterocycle," refers to a
monocyclic, bicyclic, or polycyclic ring system having from 3 to 14
ring atoms (also referred to as ring members) wherein at least one
ring atom in at least one ring is a heteroatom selected from N, O,
P, or S (and all combinations and subcombinations of ranges and
specific numbers of carbon atoms and heteroatoms therein). The
heterocycle can have from 1 to 4 ring heteroatoms independently
selected from N, O, P, or S. One or more N, C, or S atoms in a
heterocycle can be oxidized. A monocylic heterocycle preferably has
3 to 7 ring members (e.g., 2 to 6 carbon atoms and 1 to 3
heteroatoms independently selected from N, O, P, or S), and a
bicyclic heterocycle preferably has 5 to 10 ring members (e.g., 4
to 9 carbon atoms and 1 to 3 heteroatoms independently selected
from N, O, P, or S). The ring that includes the heteroatom can be
aromatic or non-aromatic. Unless otherwise noted, the heterocycle
is attached to its pendant group at any heteroatom or carbon atom
that results in a stable structure.
[0106] Heterocycles are described in Paquette, "Principles of
Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968),
particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of
Heterocyclic Compounds, A series of Monographs" (John Wiley &
Sons, New York, 1950 to present), in particular Volumes 13, 14, 16,
19, and 28; and J. Am. Chem. Soc. 82:5566 (1960).
[0107] Unless otherwise noted, the term "heterocyclo" refers to an
optionally substituted heterocycle group as defined above that is
divalent (i.e., derived by the removal of two hydrogen atoms from
the same or two different carbon atoms of a parent heterocyclic
ring system).
[0108] Examples of "heterocycle" groups include by way of example
and not limitation pyridyl, dihydropyridyl, tetrahydropyridyl
(piperidyl), thiazolyl, pyrimidinyl, furanyl, thienyl, pyrrolyl,
pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl,
indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl,
piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl,
pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,
tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,
tetrahydroisoquinolinyl, decahydroquinolinyl,
octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,
2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,
isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,
isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,
isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl,
phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,
cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,
.beta.-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl,
phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,
phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl,
imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl,
isoindolinyl, quinuclidinyl, morpholinyl, oxazolidinyl,
benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl, and
isatinoyl. Preferred "heterocycle" groups include, but are not
limited to, benzofuranyl, benzothiophenyl, indolyl, benzopyrazolyl,
coumarinyl, isoquinolinyl, pyrrolyl, thiophenyl, furanyl,
thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl,
pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl,
isothiazolyl, isoxazolyl and tetrazolyl.
[0109] A heterocycle group, whether alone or as part of another
group, can be optionally substituted with one or more groups,
preferably 1 to 2 groups, including but not limited to, optionally
substituted --C.sub.1-C.sub.8 alkyl, optionally substituted
--C.sub.2-C.sub.8 alkenyl, optionally substituted --C.sub.2-C.sub.8
alkynyl, halogen, optionally substituted --O--(C.sub.1-C.sub.8
alkyl), optionally substituted --O--(C.sub.2-C.sub.8 alkenyl),
optionally substituted --O--(C.sub.2-C.sub.8 alkynyl), optionally
substituted -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R',
--S(O).sub.2R', --S(O)R', --OH, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl and
wherein said optionally substituted O--(C.sub.1-C.sub.8 alkyl),
optionally substituted --O--(C.sub.2-C.sub.8 alkenyl), optionally
substituted --O--(C.sub.2-C.sub.8 alkynyl), optionally substituted
--C.sub.1-C.sub.8 alkyl, optionally substituted --C.sub.2-C.sub.8
alkenyl, optionally substituted --C.sub.2-C.sub.8 alkynyl, and
optionally substituted aryl groups can be further optionally
substituted with one or more substituents including, but not
limited to, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl.
[0110] By way of example and not limitation, carbon-bonded
heterocycles can be bonded at the following positions: position 2,
3, 4, 5, or 6 of a pyridine; position 3, 4, 5, or 6 of a
pyridazine; position 2, 4, 5, or 6 of a pyrimidine; position 2, 3,
5, or 6 of a pyrazine; position 2, 3, 4, or 5 of a furan,
tetrahydrofuran, thiofuran, thiophene, pyrrole or
tetrahydropyrrole; position 2, 4, or 5 of an oxazole, imidazole or
thiazole; position 3, 4, or 5 of an isoxazole, pyrazole, or
isothiazole; position 2 or 3 of an aziridine; position 2, 3, or 4
of an azetidine; position 2, 3, 4, 5, 6, 7, or 8 of a quinoline; or
position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more
typically, carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,
4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,
5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,
5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,
5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or
5-thiazolyl.
[0111] By way of example and not limitation, nitrogen bonded
heterocycles can be bonded at position 1 of an aziridine,
azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline,
imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole,
pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine,
indole, indoline, or 1H-indazole; position 2 of a isoindole, or
isoindoline; position 4 of a morpholine; and position 9 of a
carbazole, or .beta.-carboline. Still more typically, nitrogen
bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,
1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
[0112] Unless otherwise noted, the term "carbocycle," refers to a
saturated or unsaturated non-aromatic monocyclic, bicyclic, or
polycyclic ring system having from 3 to 14 ring atoms (and all
combinations and subcombinations of ranges and specific numbers of
carbon atoms therein) wherein all of the ring atoms are carbon
atoms. Monocyclic carbocycles preferably have 3 to 6 ring atoms,
still more preferably 5 or 6 ring atoms. Bicyclic carbocycles
preferably have 7 to 12 ring atoms, e.g., arranged as a bicyclo
[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10 ring atoms arranged
as a bicyclo [5,6] or [6,6] system. The term "carbocycle" includes,
for example, a monocyclic carbocycle ring fused to an aryl ring
(e.g., a monocyclic carbocycle ring fused to a benzene ring).
Carbocyles preferably have 3 to 8 carbon ring atoms.
[0113] Carbocycle groups, whether alone or as part of another
group, can be optionally substituted with, for example, one or more
groups, preferably 1 or 2 groups (and any additional substituents
selected from halogen), including, but not limited to, halogen,
optionally substituted C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl and
wherein said optionally substituted --C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), and optionally substituted aryl
groups can be further optionally substituted with one or more
substituents including, but not limited to, C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, halogen,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'', --OC(O)R'',
--C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'', --C(O)N(R'').sub.2,
--NHC(O)R'', --SR'', --SO.sub.3R'', --S(O).sub.2R'', --S(O)R'',
--OH, --N.sub.3, --NH.sub.2, --NH(R''), --N(R'').sub.2 and --CN,
where each R'' is independently selected from H, --C.sub.1-C.sub.8
alkyl, --C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, or
aryl.
[0114] Examples of monocyclic carbocylic substituents include
cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl,
1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,
1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,
cycloheptyl, and cyclooctyl. -1,3-cyclohexadienyl,
-1,4-cyclohexadienyl, -1,3-cycloheptadienyl,
-1,3,5-cycloheptatrienyl, and -cyclooctadienyl.
[0115] A "carbocyclo," whether used alone or as part of another
group, refers to an optionally substituted carbocycle group as
defined above that is divalent (i.e., derived by the removal of two
hydrogen atoms from the same or two different carbon atoms of a
parent carbocyclic ring system).
[0116] When any variable occurs more than one time in any
constituent or in any formula, its definition in each occurrence is
independent of its definition at every other. Combinations of
substituents and/or variables are permissible only if such
combinations result in stable compounds.
[0117] Unless otherwise indicated by context, a hyphen (-)
designates the point of attachment to the pendant molecule.
Accordingly, the term "--(C.sub.1-C.sub.8 alkylene)aryl" or
"--C.sub.1-C.sub.8 alkylene(aryl)" refers to a C.sub.1-C.sub.8
alkylene radical as defined herein wherein the alkylene radical is
attached to the pendant molecule at any of the carbon atoms of the
alkylene radical and one of the hydrogen atom bonded to a carbon
atom of the alkylene radical is replaced with an aryl radical as
defined herein.
[0118] When a particular group is "substituted", that group may
have one or more substituents, preferably from one to five
substituents, more preferably from one to three substituents, most
preferably from one to two substituents, independently selected
from the list of substituents. The group can, however, generally
have any number of substituents selected from halogen. Groups that
are substituted are so indicated.
[0119] It is intended that the definition of any substituent or
variable at a particular location in a molecule be independent of
its definitions elsewhere in that molecule. It is understood that
substituents and substitution patterns on the compounds of this
invention can be selected by one of ordinary skill in the art to
provide compounds that are chemically stable and that can be
readily synthesized by techniques known in the art as well as those
methods set forth herein.
[0120] Protective groups as used herein refer to groups which
selectively block, either temporarily or permanently, one reactive
site in a multifunctional compound. Suitable hydroxy-protecting
groups for use in the present invention can be administered to a
subject in the context of the present invention and may or may not
need to be cleaved from the parent compound after administration to
a subject in order for the compound to be active. Cleavage is
through normal metabolic processes within the body. Hydroxy
protecting groups are well known in the art, see, Protective Groups
in Organic Synthesis by T. W. Greene and P. G. M. Wuts (John Wiley
& sons, 3.sup.rd Edition) incorporated herein by reference in
its entirety and for all purposes and include, for example, ether
(e.g., alkyl ethers and silyl ethers including, for example,
dialkylsilylether, trialkylsilylether, dialkylalkoxysilylether),
ester, carbonate, carbamates, sulfonate, and phosphate protecting
groups. Examples of hydroxy protecting groups include, but are not
limited to, methyl ether; methoxymethyl ether, methylthiomethyl
ether, (phenyldimethylsilyl)methoxymethyl ether, benzyloxymethyl
ether, p-methoxybenzyloxymethyl ether, p-nitrobenzyloxymethyl
ether, o-nitrobenzyloxymethyl ether, (4-methoxyphenoxy)methyl
ether, guaiacolmethyl ether, t-butoxymethyl ether,
4-pentenyloxymethyl ether, siloxymethyl ether,
2-methoxyethoxymethyl ether, 2,2,2-trichloroethoxymethyl ether,
bis(2-chloroethoxy)methyl ether, 2-(trimethylsilyl)ethoxymethyl
ether, menthoxymethyl ether, tetrahydropyranyl ether,
1-methoxycylcohexyl ether, 4-methoxytetrahydrothiopyranyl ether,
4-methoxytetrahydrothiopyranyl ether S,S-Dioxide,
1-[(2-choro-4-methyl)phenyl]-4-methoxypiperidin-4-yl ether,
1-(2-fluorophneyl)-4-methoxypiperidin-4-yl ether, 1,4-dioxan-2-yl
ether, tetrahydrofuranyl ether, tetrahydrothiofuranyl ether;
substituted ethyl ethers such as 1-ethoxyethyl ether,
1-(2-chloroethoxy)ethyl ether, 1-[2-(trimethylsilyl)ethoxy]ethyl
ether, 1-methyl-1-methoxyethyl ether, 1-methyl-1-benzyloxyethyl
ether, 1-methyl-1-benzyloxy-2-fluoroethyl ether,
1-mehtyl-1phenoxyethyl ether, 2-trimethylsilyl ether, t-butyl
ether, allyl ether, propargyl ethers, p-chlorophenyl ether,
p-methoxyphenyl ether, benzyl ether, p-methoxybenzyl ether
3,4-dimethoxybenzyl ether, trimethylsilyl ether, triethylsilyl
ether, tripropylsilylether, dimethylisopropylsilyl ether,
diethylisopropylsilyl ether, dimethylhexylsilyl ether,
t-butyldimethylsilyl ether, diphenylmethylsilyl ether,
benzoylformate ester, acetate ester, chloroacetate ester,
dichloroacetate ester, trichloroacetate ester, trifluoroacetate
ester, methoxyacetate ester, triphneylmethoxyacetate ester,
phenylacetate ester, benzoate ester, alkyl methyl carbonate, alkyl
9-fluorenylmethyl carbonate, alkyl ethyl carbonate, alkyl
2,2,2,-trichloroethyl carbonate, 1,1,-dimethyl-2,2,2-trichloroethyl
carbonate, alkylsulfonate, methanesulfonate, benzylsulfonate,
tosylate, methylene acetal, ethylidene acetal, and
t-butylmethylidene ketal. Preferred protecting groups are
represented by the formulas --R.sup.a,
--Si(R.sup.a)(R.sup.a)(R.sup.a), --C(O)R.sup.a, --C(O)OR.sup.a,
--C(O)NH(R.sup.a), --S(O).sub.2R.sup.a, --S(O).sub.2OH,
P(O)(OH).sub.2, and --P(O)(OH)OR.sup.a, wherein R.sup.a is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl, --C.sub.1-C.sub.20 alkylene(carbocycle),
--C.sub.2-C.sub.20 alkenylene(carbocycle), --C.sub.2-C.sub.20
alkynylene(carbocycle), C.sub.6-C.sub.10 aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), --C.sub.1-C.sub.20
alkylene(heterocycle), --C.sub.2-C.sub.20 alkenylene(heterocycle),
or --C.sub.2-C.sub.20 alkynylene(heterocycle) wherein said alkyl,
alkenyl, alkynyl, alkylene, alkenylene, and alkynylene radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A1,
said carbocycle radicals whether alone or as part of another group
are optionally substituted with one or more groups independently
selected from A2, said aryl radicals whether alone or as part of
another group are optionally substituted with one or more groups
independently selected from A3, and said heterocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A4.
A1, A2, A3, and A4 are as defined herein.
[0121] The abbreviation "AFP" refers to
dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylened-
iamine (see Formula XVI infra).
[0122] The abbreviation "MMAE" refers to monomethyl auristatin E
(see Formula XI infra).
[0123] The abbreviation "AEB" refers to an ester produced by
reacting auristatin E with paraacetyl benzoic acid (see Formula XX
infra)
[0124] The abbreviation "AEVB" refers to an ester produced by
reacting auristatin E with benzoylvaleric acid (see Formula XXI
infra).
[0125] The abbreviation "MMAF" refers to
dovaline-valine-dolaisoleuine-dolaproine-phenylalanine (see Formula
IVIV infra).
CD19 Binding Agents
[0126] The methods described herein encompass the use of CD19
binding agents and ligand-drug conjugate compounds wherein the
ligand unit is an anti-CD19 binding agent that specifically binds
to CD19. The CD19 binding agent can be, for example, an anti-CD19
antibody, an anti-CD19 antigen-binding fragment, or other CD19
binding agent comprising the amino acid sequence of a humanized
antibody heavy and/or light chain variable region, or derivative
thereof.
[0127] In certain aspects, the anti-CD19 binding agents of the
present invention include a heavy and/or light chain variable
domain, the heavy and light chain variable domains each have (a) a
set of three CDRs identical or substantially identical to the
corresponding CDRs of mAb mBU12, and (b) a set of four variable
region framework regions identical or substantially identical to
framework regions from a human immunoglobulin.
[0128] The present invention encompasses embodiments wherein the
framework regions chosen for the heavy chain variable region of the
CD19 binding agents of the present invention are the human germline
V.sub.H exons V.sub.H2-70 or V.sub.H4-31 and the human germline
J.sub.H4 exon for the humanized FR4 sequence. In some embodiments,
the human germline J.sub.H1, J.sub.H2, J.sub.H3, J.sub.H5, or
J.sub.H6 exon is used in place of the human germline J.sub.H4 exon
for the humanized FR4 sequence.
[0129] The present invention encompasses embodiments wherein the
framework regions chosen for the light chain variable region of the
CD19 binding agents of the present invention are the human germline
V.sub.L exons V.sub.L-L6 or V.sub.LA10 and the human germline
J.sub.k2 exon for the humanized FR4 sequence. In some embodiments,
the human germline J.sub.k1, J.sub.k3, J.sub.k4, or J.sub.k5 exon
is used in place of the human germline J.sub.k2 exon for the
humanized FR4 sequence.
[0130] The present invention encompasses embodiments wherein mouse
donor residues are reintroduced into the sequence of the framework
region of the CD19 binding agents. Such residues can include, for
example, reintroduction of the mouse donor residue at one or more
of positions 75, 79, 81, 82, 82A, 82B, 82C and 89, according to the
Kabat numbering system, of the V.sub.H2-70/J.sub.H4 germline,
positions 24, 27, 29, 71, 75, 78, 79, and 89, according to the
Kabat numbering system, of the V.sub.H4-31/J.sub.H4 germline,
positions 2, 40, 41, 42, 69, 70, 71, 72, and 83, according to the
Kabat numbering system, of the V.sub.L-L6/J.sub.k2 germline, and
positions 2 and 71, according to the Kabat numbering system, of the
V.sub.LA10/J.sub.k2 germline. Additional mouse donor residues at
alternate positions can be reintroduced into the sequences.
[0131] The present invention emcompasses embodiment wherein the
CD19 binding agents described herein have amino acid sequence
modification(s) in the acceptor human germline exon in addition to
the reintroduction of mouse donor residues as well as amino acid
sequence modification(s) in the hypervariable regions. For example,
it may be desirable to improve the binding affinity and/or other
biological properties of the antibody. Amino acid sequence variants
of the CD19 binding agents are prepared by introducing appropriate
nucleotide changes into the antibody nucleic acid, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of, residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution is made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics. Substitutions may be conservative or
non-conservative substitutions. The amino acid changes also may
alter post-translational processes of the antibody, such as
changing the number or position of glycosylation sites.
[0132] A useful method for identification of certain residues or
regions of the CD19 binding agent that are favored locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed variants
are screened for the desired activity.
[0133] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions as well as intrasequence insertions of
single or multiple amino acid residues. Another type of variant is
an amino acid substitution variant. These variants have at least
one amino acid residue in the antibody molecule replaced by a
different residue. The sites of greatest interest for
substitutional mutagenesis include the hypervariable regions, but
FR alterations are also contemplated.
[0134] Substantial modifications in the biological properties of
the CD19 binding agent are accomplished by selecting substitutions
that differ significantly in their effect on maintaining (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain. Naturally-occurring residues are
divided into groups based on common side-chain properties:
[0135] (1) hydrophobic: met, ala, val, leu, ile;
[0136] (2) neutral hydrophilic: cys, ser, thr;
[0137] (3) acidic: asp, glu;
[0138] (4) basic: asn, gln, his, lys, arg;
[0139] (5) residues that influence chain orientation: gly, pro;
and
[0140] (6) aromatic: trp, tyr, phe.
[0141] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Conservative
substitutions will entail exchanging members of the same class.
[0142] One type of substitutional variant involves substituting one
or more hypervariable region residues. In some embodiments, the
resulting variant(s) selected for further development will have
improved biological properties relative to the parent binding agent
from which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the binding agent and the antigen.
Such contact residues and neighboring residues are candidates for
substitution according to the techniques elaborated herein. Once
such variants are generated, the panel of variants is subjected to
screening as described herein and binding agents with superior
properties in one or more relevant assays may be selected for
further development.
[0143] The antibodies or derivatives thereof or other binding
agents can have modifications (e.g., substitutions, deletions or
additions) in amino acid residues that interact with Fc.gamma.
receptors. In particular, antibodies or derivatives thereof or
other binding agents include antibodies or derivatives thereof or
other binding agents having modifications in amino acid residues
identified as involved in the binding interaction between the Fc
domain and one or more Fc.gamma. receptors (see infra), as well as
antibodies or derivatives thereof or other binding agents having
modifications in amino acid residues identified as involved in the
interaction between the anti-Fc domain and the FcRn receptor (see,
e.g., International Publication No. WO 97/34631, which is
incorporated herein by reference in its entirety).
[0144] In some embodiments, the binding of a target binding agent
to one or more Fc.gamma. receptors can be impaired using one or
more antibody engineering approaches known in the art. In some
embodiments, the binding of a target binding agent to one or more
Fc.gamma. receptors can be impaired by reducing the target binding
agent's effector functions using one or more antibody engineering
approaches known in the art. Illustrative, non-limiting examples
for such approaches are provided below.
[0145] Fc.gamma. receptor binding is mediated through the
interaction of a region of an antibody with an Fc gamma (Fc.gamma.)
receptor (Fc.gamma.R). The Fc region or domain refers to the
region(s) of an antibody constant region (e.g., IgG1, IgG2, IgG3,
or IgG4) that is involved in the binding interaction of the Fc
region to one or more Fc.gamma. receptors (e.g., Fc.gamma.RI
(CD64), Fc.gamma.RIIb (CD32b) or Fc.gamma.RIIIa (CD16). Both the
glycosylation status and primary amino acid sequence of the IgG Fc
region have functional effects on the Fc region-Fc.gamma.R
interaction.
[0146] Substitution of particular amino acid positions in the Fc
region of IgG isotype constant regions are known to have functional
effects on the ability of an antibody to bind to one or more
Fc.gamma. receptors. See, e.g., Shields et al., 2001, J. Biol.
Chem. 276:6591-6604, and Canfield and Morrison, 1991, J. Exp. Med.
173:1483-1491. The Fc region includes, for example and not for
limitation, amino acid residues in the hinge region and the
C.sub.H2 domain. Substitution of one or more amino acid residues in
the Fc region or portion of an IgG constant region with
non-conservative amino acids can be expected to alter, i.e., reduce
the affinity of the Fc region-Fc.gamma.R interaction. Methods for
introducing non-conservative amino acid substitutions in an
antibody or derivative thereof or other binding agent are well
known in the art.
[0147] Alternatively or additionally, cysteine residue(s) may be
introduced in or in proximity to the Fc region or portion of an IgG
constant region, thereby allowing interchain disulfide bond
formation in this region. Such interchain disulfide bond formation
can be expected to cause steric hindrance, thereby reducing the
affinity of the Fc region-Fc.gamma.R binding interaction. The
cysteine residue(s) introduced in or in proximity to the Fc region
of an IgG constant region may also serve as sites for conjugation
to therapeutic agents (i.e., coupling cytotoxic drugs using thiol
specific reagents such as maleimide derivatives of drugs). The
presence of a therapeutic agent can be expected to cause steric
hindrance, thereby reducing the affinity of the Fc
region-Fc.gamma.R binding interaction. Methods for introducing
cysteine residues in an antibody or derivative thereof or other
binding agent are well known in the art.
[0148] Alternatively or additionally, one or more N-linked
glycosylation sites may be introduced in or in proximity to the Fc
region of an IgG constant region, thereby allowing
post-translational glycosylation in this region. Such N-linked
glycosylation can be expected to cause steric hindrance, thereby
reducing the affinity of the Fc region-Fc.gamma.R binding
interaction. Methods for introducing N-linked glycosylation sites
in an antibody or derivative thereof or other binding agent are
well known in the art.
[0149] A systemic substitution of solvent-exposed amino acids of
human IgG1 Fc region has generated IgG derivatives with altered
Fc.gamma.R binding affinities (Shields et al., 2001, J. Biol. Chem.
276:6591-604). For example, when compared to parental IgG1, a
subset of these derivatives involving substitutions at
Thr256/Ser298, Ser298/Glu333, Ser298/Lys334, or
Ser298/Glu333/Lys334 to Ala demonstrate increases in both binding
affinity toward Fc.gamma.R and ADCC activity (Shields et al., 2001,
J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol.
336:1239-49). In contrast, when compared to parental IgG1, a subset
of these derivatives involving substitutions at Glu233 to
Pro/Leu234 to Val/Leu235 to Ala and Gly 236 deletion, Pro238 to
Ala, Asp265 to Ala, Asn297 to Ala, Ala 327 to Gln, or Pro329 to Ala
demonstrate decreases in binding affinities to all Fc.gamma.R; the
Asp265 to Ala substitution also resulted in decreased ADCC activity
(Shields et al., 2001, J. Biol. Chem. 276:6591-604). Amino acids in
the hinge region and the C.sub.H2 domain have been shown to
contribute to high affinity of human IgG for Fc.gamma.R (Canfield
and Morrison, 1991, J. Exp. Med. 173:1483-1491). These amino acid
positions, or amino acids in proximity thereto, involved in
mediating the Fc region-Fc.gamma.R binding interaction are
potential targets for replacement by non-conservative amino acids
and/or introduction of one or more cysteines, and/or introduction
of one or more N-linked glycosylation sites.
[0150] The in vivo half-life of an antibody can also impact on its
effector functions. In some embodiments, it is desirable to
increase the half-life of an antibody to modify its therapeutic
activities. In some embodiments, it is desirable to decrease the
half-life of an antibody to modify its therapeutic activities. FcRn
is a receptor that is structurally similar to MHC Class I antigen
that non-covalently associates with .beta.2-microglobulin. FcRn
regulates the catabolism of IgGs and their transcytosis across
tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766;
Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The IgG-FcRn
interaction takes place at pH 6.0 (pH of intracellular vesicles)
but not at pH 7.4 (pH of blood); this interaction enables IgGs to
be recycled back to the circulation (Ghetie and Ward, 2000, Ann.
Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res.
25:97-113). The region on human IgG.sub.1 involved in FcRn binding
has been mapped (Shields et al., 2001, J. Biol. Chem.
276:6591-604). Alanine substitutions at positions Pro238, Thr256,
Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human
IgG.sub.1 enhance FcRn binding (Shields et al., 2001, J. Biol.
Chem. 276:6591-604). IgG.sub.1 molecules harboring these
substitutions are expected to have longer serum half-lives.
Consequently, these modified IgG.sub.1 molecules may be able to
carry out their effector functions, and hence exert their
therapeutic efficacies, over a longer period of time compared to
unmodified IgG.sub.1.
[0151] In an embodiment, the binding agents of the present
invention having impaired binding to one or more Fc.gamma.R retain,
at least to some extent, the ability to bind FcRn. In an
embodiment, the binding agents, which have impaired binding to one
or more Fc.gamma.R, retain the ability to bind FcRn. The ability of
an antibody or derivative thereof or other binding agent to bind to
FcRn can be measured by techniques known in the art (e.g., Shields
et al., 2001, J. Biol. Chem. 276:6591-604).
[0152] A CD19 binding agent modified with respect to effector
function may, in some embodiments, have improved internalization
capability and/or increased complement-mediated cell killing and
antibody-dependent cellular cytotoxicity (ADCC). See Caron et al.
J. Exp Med. 176:1191-1195 (1992) and Shopes, B. J. Immunol.
148:2918-2922 (1992). Homodimeric antibodies with enhanced
anti-tumor activity may also be prepared using heterobifunctional
cross-linkers as described in Wolff et al. Cancer Research
53:2560-2565 (1993). Alternatively, an antibody can be engineered
which has dual Fc regions and may thereby have enhanced complement
lysis and ADCC capabilities. See Stevenson et al. Anti-Cancer Drug
Design 3:219-230 (1989).
[0153] In certain embodiments, cysteine residue(s) may be
introduced in the Fc region in order to affect the binding
interaction of the Fc region with the Fc.gamma.RIIIa receptor. In
some embodiments, an amino acid substitution of the native amino
acid to a cysteine residue is introduced at amino acid position
239, 265, 269 or 327, according to the Kabat numbering system. In
some embodiments, an amino acid substitution of the native amino
acid to a cysteine residue is introduced at amino acid position 239
or 269, according to the Kabat numbering system. In some
embodiments, an amino acid substitution of the native amino acid to
a cysteine residue is introduced at amino acid position 239,
according to the Kabat numbering system. In some embodiments, an
amino acid substitution of the native amino acid to a cysteine
residue is introduced at amino acid position 265, according to the
Kabat numbering system. In some embodiments, an amino acid
substitution of the native amino acid to a cysteine residue is
introduced at amino acid position 269, according to the Kabat
numbering system. In some embodiments, an amino acid substitution
of the native amino acid to a cysteine residue is introduced at
amino acid position 327, according to the Kabat numbering
system.
[0154] In other embodiments, an amino acid substitution of the
native amino acid to a cysteine residue is introduced at amino acid
position 236 or 238, according to the Kabat numbering system. In
some embodiments, an amino acid substitution of the native amino
acid to a cysteine residue is introduced at amino acid position
236, according to the Kabat numbering system. In some embodiments,
an amino acid substitution of the native amino acid to a cysteine
residue is introduced at amino acid position 238, according to the
Kabat numbering system.
[0155] In other embodiments, an amino acid substitution of the
native amino acid to a cysteine residue is introduced at amino acid
position 234, 235, 237, 267, 298, 299, 326, 330, or 332, according
to the Kabat numbering system. In other embodiments, an amino acid
substitution of the native amino acid to a cysteine residue is
introduced at amino acid position 237, 298, 299, 326, 330, or 332,
according to the Kabat numbering system. In other embodiments, an
amino acid substitution of the native amino acid to a cysteine
residue is introduced at amino acid position 298, 299, 326 or 330,
according to the Kabat numbering system. In some embodiments, an
amino acid substitution of the native amino acid to a cysteine
residue is introduced at amino acid position 234, according to the
Kabat numbering system. In some embodiments, an amino acid
substitution of the native amino acid to a cysteine residue is
introduced at amino acid position 235, according to the Kabat
numbering system. In some embodiments, an amino acid substitution
of the native amino acid to a cysteine residue is introduced at
amino acid position 237, according to the Kabat numbering system.
In some embodiments, an amino acid substitution of the native amino
acid to a cysteine residue is introduced at amino acid position
267, according to the Kabat numbering system. In some embodiments,
an amino acid substitution of the native amino acid to a cysteine
residue is introduced at amino acid position 298, according to the
Kabat numbering system. In some embodiments, an amino acid
substitution of the native amino acid to a cysteine residue is
introduced at amino acid position 299, according to the Kabat
numbering system. In some embodiments, an amino acid substitution
of the native amino acid to a cysteine residue is introduced at
amino acid position 326, according to the Kabat numbering system.
In some embodiments, an amino acid substitution of the native amino
acid to a cysteine residue is introduced at amino acid position
330, according to the Kabat numbering system. In some embodiments,
an amino acid substitution of the native amino acid to a cysteine
residue is introduced at amino acid position 332, according to the
Kabat numbering system.
[0156] In some embodiments, to further increase the serum half life
of the antibody or derivative thereof or other binding agent, one
may modify any salvage receptor binding epitope in the antibody or
derivative thereof or other binding agent as described in U.S. Pat.
No. 5,739,277, for example. As used herein, the term "salvage
receptor binding epitope" refers to an epitope of the Fc region of
an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is
responsible for increasing the in vivo serum half-life of the IgG
molecule. Alternatively, the serum half-life of the antibody or
derivative thereof or other binding agent may be increased by
modifying the Fc region of an antibody (e.g., IgG constant domain)
with respect to binding to Fc gamma (Fc.gamma.) receptors, as
described infra.
[0157] Antibodies may be glycosylated at conserved positions in
their constant regions (see, e.g., Jefferis and Lund, 1997, Chem.
Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32).
The oligosaccharide side chains of the immunoglobulins can affect
the protein's function (see, e.g., Boyd et al., 1996, Mol. Immunol.
32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180), and
the intramolecular interaction between portions of the glycoprotein
which can affect the conformation and presented three-dimensional
surface of the glycoprotein (see, e.g., Jefferis and Lund, supra;
Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416).
Oligosaccharides may also serve to target a given glycoprotein to
certain molecules based upon specific recognition structures. For
example, it has been reported that in agalactosylated IgG, the
oligosaccharide moiety `flips` out of the inter-C.sub.H2 space and
terminal N-acetylglucosamine residues become available to bind
mannose binding protein (see, e.g., Malhotra et al., 1995, Nature
Med. 1:237-243). Removal by glycopeptidase of the oligosaccharides
from CAMPATH-1H (a recombinant humanized murine monoclonal IgG1
antibody which recognizes the CDw52 antigen of human lymphocytes)
produced in Chinese Hamster Ovary (CHO) cells resulted in a
complete reduction in complement mediated lysis (CMCL) (Boyd et
al., 1996, Mol. Immunol. 32:1311-1318), while selective removal of
sialic acid residues using neuraminidase resulted in no loss of
DMCL. Glycosylation of antibodies has also been reported to affect
ADCC. In particular, CHO cells with tetracycline-regulated
expression of .beta.(1,4)-N-acetylglucosaminyltransferase III
(GnTIII), a glycosyltransferase catalyzing formation of bisecting
GlcNAc, was reported to have improved ADCC activity (see, e.g.,
Umana et al., 1999, Mature Biotech. 17:176-180).
[0158] Glycosylation of antibodies is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine may also be used.
[0159] Glycosylation derivatives of antibodies are derivatives in
which the glycosylation pattern of an antibody is altered. Certain
antibodies of the present invention have altered glycosylation
patterns. By altering is meant deleting one or more carbohydrate
moieties found in the antibody, adding one or more carbohydrate
moieties to the antibody, changing the composition of glycosylation
(i.e., glycosylation pattern), the extent of glycosylation, or the
like. In certain embodiments, the antibodies of the present
invention have reduced core fucosylation.
[0160] Addition of glycosylation sites to the antibody can be
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tripeptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the sequence of the original
antibody (for O-linked glycosylation sites). Similarly, removal of
glycosylation sites can be accomplished by amino acid alteration
within the native glycosylation sites of the antibody.
[0161] The amino acid sequence is usually altered by altering the
underlying nucleic acid sequence. These methods include, but are
not limited to, isolation from a natural source (in the case of
naturally-occurring amino acid sequence derivatives) or preparation
by oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, or cassette mutagenesis of an earlier prepared
derivative or a non-derivative version of the antibody.
[0162] The glycosylation (including glycosylation pattern) of
antibodies may also be altered without altering the amino acid
sequence or the underlying nucleotide sequence. Glycosylation
largely depends on the host cell used to express the antibody.
Since the cell type used for expression of recombinant
glycoproteins, e.g., antibodies, as potential therapeutics is
rarely the native cell, significant variations in the glycosylation
pattern of the antibodies can be expected. See, e.g., Hse et al.,
1997, J. Biol. Chem. 272:9062-9070. In addition to the choice of
host cells, factors which affect glycosylation during recombinant
production of antibodies include growth mode, media formulation,
culture density, oxygenation, pH, purification schemes, and the
like. Various methods have been proposed to alter the glycosylation
pattern achieved in a particular host organism, including
introducing or overexpressing certain enzymes involved in
oligosaccharide production (see, e.g., U.S. Pat. Nos. 5,047,335;
5,510,261; and 5,278,299). Glycosylation, or certain types of
glycosylation, can be enzymatically removed from the glycoprotein,
for example using endoglycosidase H (Endo H). In addition, the
recombinant host cell can be genetically engineered, e.g., made
defective in processing certain types of polysaccharides. These and
similar techniques are well known in the art.
[0163] The glycosylation structure of antibodies can be readily
analyzed by conventional techniques of carbohydrate analysis,
including lectin chromatography, NMR, mass spectrometry, HPLC, GPC,
monosaccharide compositional analysis, sequential enzymatic
digestion, and HPAEC-PAD, which uses high pH anion exchange
chromatography to separate oligosaccharides based on charge.
Methods for releasing oligosaccharides for analytical purposes are
also known, and include, without limitation, enzymatic treatment
(commonly performed using peptide-N-glycosidase
F/endo-.beta.-galactosidase), elimination using harsh alkaline
environment to release mainly O-linked structures, and chemical
methods using anhydrous hydrazine to release both N- and O-linked
oligosaccharides.
[0164] The following table provides a summary of the regions of
chimeric and humanized BU12 to which each sequence identifier (SEQ
ID NO.) corresponds.
TABLE-US-00001 Nucleotide or SEQ MOLECULE Amino Acid ID NO Leader
Sequence Amino Acid 1 (Heavy Chain Region) Heavy Chain Variable
Region Amino Acid 2 (VH2-70/J.sub.H4 germline)/Also referred to as
Variant HA Heavy Chain Constant domain Amino Acid 3 (IgG.sub.1)
Variant HB Amino Acid 4 Heavy Chain Variable Region
(VH2-70/J.sub.H4 germline) Variant HC Amino Acid 5 Heavy Chain
Variable Region (VH2-70/J.sub.H4 germline) Variant HD Amino Acid 6
Heavy Chain Variable Region (VH2-70/J.sub.H4 germline) Variant HE
Amino Acid 7 Heavy Chain Variable Region (VH2-70/J.sub.H4 germline)
Heavy Chain Variable Region Amino Acid 8 (Murine) Heavy Chain
Variable Region Amino Acid 9 (VH4-31/J.sub.H4 germline)/also
referred to as Variant HF Variant HG Amino Acid 10 Heavy Chain
Variable Region (VH4-31/J.sub.H4 germline) Variant HH Amino Acid 11
Heavy Chain Variable Region (VH4-31/J.sub.H4 germline) Variant HI
Amino Acid 12 Heavy Chain Variable Region (VH4-31/J.sub.H4
germline) Variant HJ Amino Acid 13 Heavy Chain Variable Region
(VH4-31/J.sub.H4 germline) Variant HK Amino Acid 14 Heavy Chain
Variable Region (VH4-31/J.sub.H4 germline) Variant HL Amino Acid 15
Heavy Chain Variable Region (VH4-31/J.sub.H4 germline) Leader
Sequence Amino Acid 16 (Light Chain Region) Light Chain Variable
Region Amino Acid 17 (VL-L6/J.sub.k2 germline)/Also referred to as
Variant LA Light Chain Constant domain Amino Acid 18 (Kappa domain)
Variant LB Amino Acid 19 Light Chain Variable Region
(VL-L6/J.sub.k2 germline) Variant LC Amino Acid 20 Light Chain
Variable Region (VL-L6/J.sub.k2 germline) Variant LD Amino Acid 21
Light Chain Variable Region (VL-L6/J.sub.k2 germline) Variant LE
Amino Acid 22 Light Chain Variable Region (VL-L6/J.sub.k2 germline)
Variant LF Amino Acid 23 Light Chain Variable Region
(VL-L6/J.sub.k2 germline) Variant LG Amino Acid 24 Light Chain
Variable Region (VL-L6/J.sub.k2 germline) Light Chain Variable
Region Amino Acid 25 (Murine) Light Chain Variable Region Amino
Acid 26 (VL-A10/J.sub.k2 germline)/Also referred to as Variant LH
domain Variant LI Amino Acid 27 Light Chain Variable Region
(VL-A10/J.sub.k2 germline) Consensus sequence for Heavy Amino Acid
28 Chain Variable Region (VH2-70/J.sub.H4 germline) Consensus
sequence for Heavy Amino Acid 29 Chain Variable Region
(VH4-31/J.sub.H4 germline) Consensus sequence for Light Amino Acid
30 Chain Variable Region (VL-L6/J.sub.k2 germline) Consensus
sequence for Light Amino Acid 31 Chain Variable Region
(VL-A10/J.sub.k2 germline) Consensus sequence for Heavy Amino Acid
32 Chain Variable Region (VH2-70/J.sub.H1-6 germline) Consensus
sequence for Heavy Amino Acid 33 Chain Variable Region
(VH4-31/J.sub.H1-6 germline) Consensus sequence for Light Amino
Acid 34 Chain Variable Region (VL-L6/J.sub.k1-5 germline) Consensus
sequence for Light Amino Acid 35 Chain Variable Region
(VL-A10/J.sub.k1-5 germline) Heavy Chain Constant Domain Amino Acid
36 (IgG.sub.2) Heavy Chain Constant Domain Amino Acid 37
(IgG.sub.3) Heavy Chain Constant Domain Amino Acid 38 (IgG.sub.4)
Heavy Chain Constant Domain Amino Acid 39 Variant
(IgG.sub.1V.sub.1) Leader Sequence Nucleotide 40 (Heavy Chain
Region) Heavy Chain Variable Region Nucleotide 41 (VH4-31/J.sub.H4
germline)/also referred to as Variant HF Heavy Chain Constant
domain Nucleotide 42 (IgG.sub.1) Leader Sequence Nucleotide 43
(Light Chain Region) Variant LG Nucleotide 44 Light Chain Variable
Region (VL-L6/J.sub.k2 germline) Light Chain Constant domain
Nucleotide 45 (Kappa domain) Heavy Chain CDR1 Amino Acid 46 Heavy
Chain CDR2 Amino Acid 47 Heavy Chain CDR3 Amino Acid 48 Light Chain
CDR1 Amino Acid 49 Light Chain CDR2 Amino Acid 50 Light Chain CDR3
Amino Acid 51
[0165] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:2. The amino acid sequence can be,
for example, the amino acid sequence of SEQ ID NO:2 having any
number of substitutions provided that the CD19 binding agent
retains functional activity (i.e., CD19 binding activity) and that
the sequence retains substantial or complete identity to the amino
acid sequence set forth in SEQ ID NO:2. Exemplary heavy chain
variable regions comprise an amino acid sequence that is identical
to the amino acid sequence set forth in SEQ ID NO:2 optionally
having at least one amino acid substitution, preferably 0, 1 or 2
amino acid substitutions, at positions 75, 79, 81, 82, 82A, 82B,
82C or 89 of the amino acid sequence set forth in SEQ ID NO:2,
according to the Kabat numbering systems. Exemplary sequences
include, for example, the amino acid sequences set forth in SEQ ID
NOs:2, 4, 5, 6, and 7. In one aspect, the CD19 binding agent that
comprises a heavy chain variable region comprising an amino acid
sequence that is identical or substantially identical (i.e., having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity) to the amino acid sequence set forth in SEQ ID NO:2
comprises the CDR regions of the antibody mBU12, i.e., SEQ ID
NO:46, 47, and 48.
[0166] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:9. The amino acid sequence can be,
for example, the amino acid sequence of SEQ ID NO:9 having any
number of substitutions provided that the CD19 binding agent
retains functional activity and that the sequence retains
substantial or complete identity to the amino acid sequence set
forth in SEQ ID NO:9. Exemplary heavy chain variable regions
comprise an amino acid sequence that is identical to the amino acid
sequence set forth in SEQ ID NO:9 optionally having at least one
amino acid substitution, preferably 0, 1 or 2 amino acid
substitutions, at positions 24, 27, 29, 71, 75, 78, 79, or 89 of
the amino acid sequence set forth in SEQ ID NO:9, according to the
Kabat numbering systems. Exemplary sequences include, for example,
the amino acid sequences set forth in SEQ ID NOs:9, 10, 11, 12, 13,
14, and 15. In one aspect, the CD19 binding agent that comprises a
heavy chain variable region comprising an amino acid sequence that
is identical or substantially identical (i.e., having at least 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to
the amino acid sequence set forth in SEQ ID NO:9 comprises the CDR
regions of antibody mBU12, i.e., SEQ ID NO:46, 47, and 48.
[0167] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:17. The amino acid sequence can be,
for example, the amino acid sequence of SEQ ID NO:17 having any
number of substitutions provided that the CD19 binding agent
retains functional activity and that the sequence retains
substantial or complete identity to the amino acid sequence set
forth in SEQ ID NO:17. Exemplary light chain variable regions
comprise an amino acid sequence that is identical to the amino acid
sequence set forth in SEQ ID NO:17 optionally having at least one
amino acid substitution, preferably 0, 1 or 2 amino acid
substitutions, at positions 2, 40, 41, 42, 69, 70, 71, 72 and 83 of
the amino acid sequence set forth in SEQ ID NO:17, according to the
Kabat numbering systems. Exemplary sequences include, for example,
the amino acid sequences set forth in SEQ ID NOs:17, 19, 20, 21,
22, 23, and 24. In one aspect, the CD19 binding agent that
comprises a light chain variable region comprising an amino acid
sequence that is identical or substantially identical (i.e., having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity) to the amino acid sequence set forth in SEQ ID NO:17
comprises the CDR regions of the antibody mBU12, i.e., SEQ ID NO:49
50, and 51.
[0168] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:26. The amino acid sequence can be,
for example, the amino acid sequence of SEQ ID NO:26 having any
number of substitutions provided that the CD19 binding agent
retains functional activity and that the sequence retains
substantial or complete identity to the amino acid sequence set
forth in SEQ ID NO:26. Exemplary light chain variable regions
comprise an amino acid sequence that is identical to the amino acid
sequence set forth in SEQ ID NO:26 optionally having at least one
amino acid substitution at positions 2 and 71 of the amino acid
sequence set forth in SEQ ID NO:26, according to the Kabat
numbering systems. Exemplary sequences include, for example, the
amino acid sequences set forth in SEQ ID NOs:26 and 27. In one
aspect, the CD19 binding agent that comprises a light chain
variable region comprising an amino acid sequence that is identical
or substantially identical (i.e., having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino
acid sequence set forth in SEQ ID NO:26 comprises the CDR regions
of the antibody mBU12, i.e., SEQ ID NO:49, 50, and 51.
[0169] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:2 as
provided above, and further comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:17, as
provided above. In any of these embodiments, the heavy chain
variable region can be joined to a constant region as set forth,
for example, in SEQ ID NOs:3, or 36-39 and the light chain variable
region can be joined to a constant region as set forth, for
example, in SEQ ID NO:18. In certain embodiments, the heavy chain
variable region will further comprise the amino acid sequence set
forth in SEQ ID NO:1 and the light chain variable region will
further comprise the amino acid sequence set forth in SEQ ID
NO:16.
[0170] Accordingly, in certain embodiments, the CD19 binding agent
will comprise a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO:2 and a light chain comprising the amino acid
sequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a
heavy chain comprising the amino acid sequence set forth in SEQ ID
NO:4 and a light chain comprising the amino acid sequence set forth
in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a heavy chain
comprising the amino acid sequence set forth in SEQ ID NO:5 and a
light chain comprising the amino acid sequence set forth in SEQ ID
NO: 17, 19, 20, 21, 22, 23 or 24; a heavy chain comprising the
amino acid sequence set forth in SEQ ID NO:6 and a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 17, 19,
20, 21, 22, 23 or 24; or a heavy chain comprising the amino acid
sequence set forth in SEQ ID NO:7 and a light chain comprising the
amino acid sequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23
or 24. In any of these embodiments, the heavy chain variable region
can be joined to a constant region as set forth, for example, in
SEQ ID NOs:3, or 36-39 and the light chain variable region can be
joined to a constant region as set forth, for example, in SEQ ID
NO:18. In certain embodiments, the heavy chain variable region will
further comprise the amino acid sequence set forth in SEQ ID NO:1
and the light chain variable region will further comprise the amino
acid sequence set forth in SEQ ID NO:16.
[0171] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:2 as provided above, and further
comprises a light chain variable region comprising an amino acid
sequence that is identical or substantially identical (i.e., having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity) to the amino acid sequence set forth in SEQ ID NO:26, as
provided above. In any of these embodiments, the heavy chain
variable region can be joined to a constant region as set forth,
for example, in SEQ ID NOs:3, or 36-39 and the light chain variable
region can be joined to a constant region as set forth, for
example, in SEQ ID NO:18. In certain embodiments, the heavy chain
variable region will further comprise the amino acid sequence set
forth in SEQ ID NO:1 and the light chain variable region will
further comprise the amino acid sequence set forth in SEQ ID
NO:16.
[0172] Accordingly, in certain embodiments, the CD19 binding agent
will comprise a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO:2 and a light chain comprising the amino acid
sequence set forth in SEQ ID NO: 26 or 27; a heavy chain comprising
the amino acid sequence set forth in SEQ ID NO:4 and a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 26 or
27; a heavy chain comprising the amino acid sequence set forth in
SEQ ID NO:5 and a light chain comprising the amino acid sequence
set forth in SEQ ID NO: 26 or 27; a heavy chain comprising the
amino acid sequence set forth in SEQ ID NO:6 and a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 26 or
27; or a heavy chain comprising the amino acid sequence set forth
in SEQ ID NO:7 and a light chain comprising the amino acid sequence
set forth in SEQ ID NO: 26 or 27. In any of these embodiments, the
heavy chain variable region can be joined to a constant region as
set forth, for example, in SEQ ID NOs:3, or 36-39 and the light
chain variable region can be joined to a constant region as set
forth, for example, in SEQ ID NO:18. In certain embodiments, the
heavy chain variable region will further comprise the amino acid
sequence set forth in SEQ ID NO:1 and the light chain variable
region will further comprise the amino acid sequence set forth in
SEQ ID NO:16.
[0173] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:9 as provided above, and further
comprises a light chain variable region comprising an amino acid
sequence that is identical or substantially identical (i.e., having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity) to SEQ ID NO:17, as provided above. In any of these
embodiments, the heavy chain variable region can be joined to a
constant region as set forth, for example, in SEQ ID NOs:3, or
36-39 and the light chain variable region can be joined to a
constant region as set forth, for example, in SEQ ID NO:18. In
certain embodiments, the heavy chain variable region will further
comprise the amino acid sequence set forth in SEQ ID NO:1 and the
light chain variable region will further comprise the amino acid
sequence set forth in SEQ ID NO:16.
[0174] Accordingly, in certain embodiments, the CD19 binding agent
will comprise a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO:9 and a light chain comprising the amino acid
sequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a
heavy chain comprising the amino acid sequence set forth in SEQ ID
NO:10 and a light chain comprising the amino acid sequence set
forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24; a heavy chain
comprising the amino acid sequence set forth in SEQ ID NO:11 and a
light chain comprising the amino acid sequence set forth in SEQ ID
NO: 17, 19, 20, 21, 22, 23 or 24; a heavy chain comprising the
amino acid sequence set forth in SEQ ID NO:12 and a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 17, 19,
20, 21, 22, 23 or 24; a heavy chain comprising the amino acid
sequence set forth in SEQ ID NO:13 and a light chain comprising the
amino acid sequence set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23
or 24 a heavy chain comprising the amino acid sequence set forth in
SEQ ID NO:14 and a light chain comprising the amino acid sequence
set forth in SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24 or a heavy
chain comprising the amino acid sequence set forth in SEQ ID NO:15
and a light chain comprising the amino acid sequence set forth in
SEQ ID NO: 17, 19, 20, 21, 22, 23 or 24. In any of these
embodiments, the heavy chain variable region can be joined to a
constant region as set forth, for example, in SEQ ID NOs:3, or
36-39 and the light chain variable region can be joined to a
constant region as set forth, for example, in SEQ ID NO:18. In
certain embodiments, the heavy chain variable region will further
comprise the amino acid sequence set forth in SEQ ID NO:1 and the
light chain variable region will further comprise the amino acid
sequence set forth in SEQ ID NO:16.
[0175] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:9 as provided above, and further
comprises a light chain variable region comprising an amino acid
sequence that is identical or substantially identical (i.e., having
at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
identity) to the amino acid sequence set forth in SEQ ID NO:26, as
provided above. In any of these embodiments, the heavy chain
variable region can be joined to a constant region as set forth,
for example, in SEQ ID NOs:3, or 36-39 and the light chain variable
region can be joined to a constant region as set forth, for
example, in SEQ ID NO:18. In certain embodiments, the heavy chain
variable region will further comprise the amino acid sequence set
forth in SEQ ID NO:1 and the light chain variable region will
further comprise the amino acid sequence set forth in SEQ ID
NO:16.
[0176] Accordingly, in certain embodiments, the CD19 binding agent
will comprise a heavy chain comprising the amino acid sequence set
forth in SEQ ID NO:9 and a light chain comprising the amino acid
sequence set forth in SEQ ID NO: 26 or 27; a heavy chain comprising
the amino acid sequence set forth in SEQ ID NO:10 and a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 26 or
27; a heavy chain comprising the amino acid sequence set forth in
SEQ ID NO:11 and a light chain comprising the amino acid sequence
set forth in SEQ ID NO: 26 or 27; a heavy chain comprising the
amino acid sequence set forth in SEQ ID NO:12 and a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 26 or
27; a heavy chain comprising the amino acid sequence set forth in
SEQ ID NO:13 and a light chain comprising the amino acid sequence
set forth in SEQ ID NO: 26 or 27; heavy chain comprising the amino
acid sequence set forth in SEQ ID NO:14 and a light chain
comprising the amino acid sequence set forth in SEQ ID NO: 26 or
27; or a heavy chain comprising the amino acid sequence set forth
in SEQ ID NO:15 and a light chain comprising the amino acid
sequence set forth in SEQ ID NO: 26 or 27. In any of these
embodiments, the heavy chain variable region can be joined to a
constant region as set forth, for example, in SEQ ID NOs:3, or
36-39 and the light chain variable region can be joined to a
constant region as set forth, for example, in SEQ ID NO:18. In
certain embodiments, the heavy chain variable region will further
comprise the amino acid sequence set forth in SEQ ID NO:1 and the
light chain variable region will further comprise the amino acid
sequence set forth in SEQ ID NO:16.
[0177] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:28 or SEQ ID NO:32. The amino acid
sequence can be, for example, the amino acid sequence of SEQ ID
NO:28 or SEQ ID NO:32 having any number of substitutions provided
that the CD19 binding agent retains functional activity (i.e., CD19
binding activity) and that the sequence retains substantial or
complete identity to the amino acid sequence set forth in SEQ ID
NO:28 or SEQ ID NO:32, respectively.
[0178] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:29 or SEQ ID NO:33. The amino acid
sequence can be, for example, the amino acid sequence of SEQ ID
NO:29 or SEQ ID NO:33 having any number of substitutions provided
that the CD19 binding agent retains functional activity and that
the sequence retains substantial or complete identity to the amino
acid sequence set forth in SEQ ID NO: 29 or SEQ ID NO:33,
respectively.
[0179] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:30 or SEQ ID NO:34. The amino acid
sequence can be, for example, the amino acid sequence of SEQ ID NO:
30 or SEQ ID NO:34 having any number of substitutions provided that
the CD19 binding agent retains functional activity and that the
sequence retains substantial or complete identity to the amino acid
sequence set forth in SEQ ID NO: 30 or SEQ ID NO:34,
respectively.
[0180] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:31 or SEQ ID NO:35. The amino acid
sequence can be, for example, the amino acid sequence of SEQ ID
NO:31 or SEQ ID NO:35 having any number of substitutions provided
that the CD19 binding agent retains functional activity and that
the sequence retains substantial or complete identity to the amino
acid sequence set forth in SEQ ID NO: 31 or SEQ ID NO:35,
respectively.
[0181] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:28 or
SEQ ID NO:32 as provided above, and further comprises a light chain
variable region comprising an amino acid sequence that is identical
or substantially identical (i.e., having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID
NO:30 or SEQ ID NO:34, as provided above.
[0182] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:28 or SEQ ID NO:32 as provided
above, and further comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:31 or SEQ ID NO:35, as provided
above.
[0183] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:29 or SEQ ID NO:33 as provided
above, and further comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:30 or
SEQ ID NO:34 as provided above.
[0184] A humanized antibody comprising a heavy chain variable
region of SEQ ID NO:9 and a light chain variable region of SEQ ID
NO:17 has all six intact CDRs from the mouse BU12 antibody and
entirely human variable region framework amino acids. In contrast
to many other humanized antibodies, such an antibody has useful
binding affinity to its antigen even without any further
substitutions. However, such an antibody also provides a starting
point for making variants. Some such variants comprise a heavy
chain variable region having at least 90% sequence identity
(spanning its entire length) to SEQ ID NO:9 and a light chain
variable region having at least 90% sequence identity to SEQ ID
NO:17. Some variants have no more than 5, 4, 3, 2 or 1 amino acids
that differ from SEQ ID NO:9 in the heavy chain variable region and
no more than 5, 4, 3, 2, or 1 amino acids that differ from SEQ ID
NO:17 in the light chain variable region.
[0185] Some variants differ from the above antibody by substitution
of one or more amino acids in the variable region framework
relative to SEQ ID NO:9 or SEQ ID NO:17. The substitution can be
with an amino acid occupying the corresponding position (unless
otherwise indicated positions are by Kabat numbering) in the heavy
or light chain variable region respectively of the BU12 antibody
(sometimes referred to as a donor antibody). Substitutions with
donor amino acids often increase the affinity of the resulting
humanized antibody for its antigen by conferring a framework
conformation in the humanized antibody more closely resembling that
in the donor mouse antibody. A donor substitution at position L83
is particularly advantageous in increasing affinity as shown in
FIG. 2 (compare light chain G with the substitution and H without
the substitution). Other positions differing between SEQ ID NO:2
and the BU12 heavy chain in which substitutions can be performed
include H3, H24, H27, H29, H41, H71, H75, H789, H79, H83 and H89 in
which the positions are occupied by Q, V, G, I, P, V, K, F, P, T
and V respectively. Light chain variable region framework positions
differing between SEQ ID NO:17 and the BU12 light chain include
positions L2, L40, L41, L42, L69, L70, L71, L72 and L83 occupied by
I, P, G, Q, T, D, F, T and F respectively in the mouse BU12 light
chain. The effect of many of these substitutions on antibody
affinity is also shown in FIG. 2. It can be seen that some of these
substitutions or combinations of substitutions increase affinity.
Some preferred heavy chain substitution include one or more of H71,
H75, H78 and H79 occupied by V, K, F and P respectively. Some
preferred light chain substitutions include one or more of
positions L2, L69, L71, L72 and L83 is occupied by I, T, F, T and F
respectively. None of the tested substitutions or combinations of
substitutions caused an unacceptable loss of affinity.
[0186] How many donor substitutions to include reflects a balance
of competing considerations. In general, substitutions which
significantly increase affinity are desirable. However, minimizing
the total number of variable region framework substitutions is also
advantageous in reducing potential immunogenicity. A humanized
antibody having no substitutions in the heavy chain variable region
framework and a L83 donor substitution of the light chain variable
region framework represents a preferred balance between maximizing
affinity and minimizing immunogenicity. Many other permutations are
possible.
[0187] As well as or instead of donor substitutions, a variable
region framework amino acid can be substituted with the amino acid
occupying the corresponding position in another human antibody
sequence or a consensus of human antibody sequences (see, .e.g.,
Queen, U.S. Pat. No. 7,022,500). The rationale for such a
substitution is often to substitute a relatively rare amino acid in
human immunoglobulin sequences with a more common amino acid for
that position with a view to reducing immunogenicity. In humanized
antibodies in which the variable region frameworks are provided by
germline sequences, such substitutions are possible but generally
not necessary because germline sequences lack rare amino acids that
may be introduced by somatic mutation.
[0188] Variable region framework amino acids can also be
substituted with amino acids that are neither donor amino acids or
consensus amino acids. Such substitutions are preferably
conservative amino acid substitutions. Although many substitutions
have little effect on affinity, they may increase immunogenicity
and thus in general are not preferred.
[0189] Substitution of one or more CDR residues or omission of one
or more CDRs is also possible. Numerous antibodies have been
described in the scientific literature in which one or two CDRs can
be dispensed with for binding. Padlan et al., FASEB Journal 9:
133-139 (1995) analyzed the contact regions between antibodies and
their antigens, based on published crystal structures, and
concluded that only about one fifth to one third of CDR residues
actually contact the antigen. Padlan et al. termed these residues
SDR (for specificity determining residues). Padlan also found many
antibodies in which one or two CDRs had no amino acids in contact
with an antigen. Likewise, Vajdos et al (Journal of Molecular
Biology, vol. 320, pp. 415-428 (2002) reported that CDR1 of the
light chain of an antibody against ErbB2 was not involved in
binding. Such teaching has been applied to antibody humanization
by, for example, Iwahashi et al., Mol. Immunol. 36:1079-1091,
(1999), who showed that they could graft only L1 and L3, or L2 and
L3, of the CR49 murine antibody onto a human framework and retain
high affinity interaction with the antigen. Similarly, Tamura et
al, Journal of Immunology, 2000, 164:1432-1441 (2000) reported that
light chain CDRs 1 and 2 could be dispensed with entirely in a
humanized anti-carcinoma antibody, as could several residues in the
remaining CDRs. The substitution of certain regions within CDRs is
based on the same principle as omitting dispensable CDRs, namely
that only a small subset of CDR residues, the SDR's, actually
contact antigen.
[0190] CDR residues not contacting antigen can be identified based
on previous studies (for example residues H60-H65 in CDRH2 are
often not required), from regions of Kabat CDRs lying outside
Chothia CDRs, by molecular modeling and/or empirically. If a CDR or
residue(s) thereof is omitted, it is usually substituted with an
amino acid occupying the corresponding position in human acceptor
sequence supplying the variable region framework sequences (in this
example, VH4-31/JH4 for the heavy chain and VL-L6 JK2 for the light
chain). The number of such substitutions to include reflects a
balance of competing considerations Such substitutions are
potentially advantageous in decreasing the number of mouse amino
acids in a humanized antibody and consequently decreasing potential
immunogenicity. However, substitutions can also cause changes of
affinity, and significant reductions in affinity are preferably
avoided. Positions for substitution within CDRs and amino acids to
substitute can also be selected empirically. Empirical
substitutions can be conservative or non-conservative
substitutions. However, in general empirical substitutions do not
have the advantage of mouse to human substitutions in reducing
immunogenicity. Empirical substitutions can increase or decrease
affinity of the resulting humanized antibody.
[0191] In general humanized antibodies with satisfactory binding
affinity to CD19 and lack of substantial immunogenicity can be
obtained by individual screening of a view variants made according
to the above principles and/or in accordance with the present
examples. However, very large numbers of variants can be
simultaneously screened using a display selection method such as
phage display (see (Dower et al., WO91/17271; McCafferty et al.,
WO92/001047; and Winter, WO92/20791). The same considerations apply
mutatis mutandis in designing variants of other humanized
antibodies or antibody chains disclosed herein. For example, SEQ ID
NO:2 provides an alternative starting point to SEQ ID NO:9 for
design of heavy chain variants. SEQ ID NO:2 comprises the three
heavy chain CDRs of the BU12 antibody with a fully human variable
region framework sequence of the VH2-70 and JH4 genes. SEQ ID NO:26
provides an alternative starting point to SEQ ID NO:17 for design
of light chain variants. SEQ ID NO:26 comprises the three light
chain CDRs of the BU12 antibody in a fully human framework sequence
of the A10 and JK2 genes. Specific heavy chain variable region
framework positions for potential substitution and the amino acids
to substitute into such positions are indicated in the table
"non-homologous FR residues BU12 v.s VH431) in Example. Specific
light chain variable region framework positions for potential
substitution in SEQ ID NO:26 are indicated in the table
"Nono-homologous FR residues BU12 VL vs. L6 and A10" in Example
2.
[0192] The present invention encompasses embodiments wherein the
CD19 binding agent comprises a heavy chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:29 or SEQ ID NO:33 as provided
above, and further comprises a light chain variable region
comprising an amino acid sequence that is identical or
substantially identical (i.e., having at least 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the amino acid
sequence set forth in SEQ ID NO:31 or SEQ ID NO:35, as provided
above.
[0193] The present invention encompasses embodiments wherein the
heavy chain variable region further comprises a leader sequence.
One such leader sequence is set forth in SEQ ID NO:1.
[0194] The present invention encompasses embodiments wherein the
light chain variable region further comprises a leader sequence.
One such leader sequence is set forth in SEQ ID NO:16.
[0195] The CD19 binding agent can optionally include an antibody
effector region. The effector domain(s) can be, for example, an Fc
region such as a hinge-C.sub.H2-C.sub.H3 region of an
immunoglobulin, or a portion or fragment of an effector region
preferably having effector function. Antigen-binding antibody
fragments, including single-chain antibodies, can comprise, for
example, the variable region(s) in combination with the entirety or
a portion of an effector region (e.g., a C.sub.H2 and/or C.sub.H3
domain alone or in combination with a C.sub.H1, hinge and/or
C.sub.L domain). Also, antigen-binding fragments can comprise any
combination of effector regions. In some embodiments, the anti-CD19
antibody can be a single chain antibody comprising a CD19-binding
variable region joined to hinge-C.sub.H2-C.sub.H3 domains.
[0196] The effector region of the anti-CD19 antibody can be from
any suitable immunoglobulin isotype. A CD19 binding agent can be
expressed as a recombinant fusion protein comprising of the
appropriate constant domains to yield the desired effector
function(s).
[0197] The present invention encompasses embodiments wherein the
heavy chain variable region of a CD19 binding agent is joined to a
constant region, such an IgG, i.e., IgG1 constant region or IgG2
constant region, or altered constant region, e.g., IgG1V1.
Exemplary constant region domains are provided as SEQ ID NO:3 and
36-39.
[0198] The present invention also encompasses embodiments wherein
the light chain variable region of a CD19 binding agent is joined
to a constant region, such as a kappa constant region. An exemplary
constant region domain is provided as SEQ ID NO:18.
[0199] The present invention encompasses embodiments wherein the
light chain variable region of a CD19 binding agent is joined to a
constant region, such as a kappa constant region. An exemplary
constant region domain is provided as SEQ ID NO:18 and the heavy
chain variable region of a CD19 binding agent is joined to a
constant region, such an IgG, i.e., IgG1 constant region or IgG2
constant region, or altered constant region, e.g., IgG1V1.
Exemplary constant region domains are provided as SEQ ID NO: 3 and
36-39.
[0200] In some embodiments, a CD19 binding agent can be a CD19
binding agent, comprising a human or non-human Fc region or portion
thereof. For example, the antibody can include an Fc region or
portion thereof of non-human origin, e.g., rodent (e.g., mouse or
rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse,
chicken or monkey (e.g., macaque, rhesus, cynomolgous or the like)
linked to humanized heavy and/or light chain variable regions.
[0201] A CD19 binding agent, such as an antibody, can be
monospecific, bispecific, trispecific, or of greater
multispecificity. Multispecific antibodies may be specific for
different epitopes of CD19 and/or may be specific for both CD19 as
well as for a heterologous protein. (See, e.g., PCT Publications WO
93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt et al.,
1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893; 4,714,681;
4,925,648; 5,573,920; and U.S. Pat. No. 5,601,819; Kostelny et al.,
1992, J. Immunol. 148:1547-1553.) Multispecific antibodies,
including bispecific and trispecific antibodies, useful for
practicing the methods described herein are antibodies that
immunospecifically bind to both CD19 and a second cell surface
receptor or receptor complex, i.e., one that mediates ADCC, ADCP,
and/or CDC.
[0202] CD19 binding agents may also be described or specified in
terms of their binding affinity to CD19. Typical binding affinities
include those with a dissociation constant or Kd less than
5.times.10.sup.-6 M, 10.sup.-6M, 5.times.10.sup.-7 M, 10.sup.-7 M,
5.times.10.sup.-8M, 10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M,
5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11 M,
10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M, 5.times..sup.-13
M, 10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15M, or 10.sup.-15 M.
[0203] The present invention encompasses nucleic acids encoding a
CD19 binding agent. The CD19 binding agent can be, for example, a
fully humanized antibody or a humanized antigen-binding fragment.
In some embodiments, the nucleic acid encodes a polypeptide chain
having the amino acid sequence or having substantial identity to
the amino acid sequences set forth in SEQ ID NOs: 2, 4, 5, 6, 7, 9,
10, 11, 12, 13, 14, 15, 17, 19, 20, 21, 22, 23, 24, 26 or 27. In
certain embodiments, a nucleic acid of the present invention will
comprise the nucleotide sequence set forth in SEQ ID NOS. 40, 41,
42, 43, 44, or 45. In certain embodiments, the nucleic acid will
encode a heavy chain variable region of an antibody and will
comprise one or more of the sequences set forth in SEQ ID NOS. 40,
41, and 42. In certain embodiments, the nucleic acid will encode a
light chain variable region of an antibody and will comprise one or
more of the sequences set forth in SEQ ID NOS. 43, 44, and 45.
[0204] Also included in some embodiments are nucleic acids encoding
a CD19 binding agent that hybridize under low, moderate, and high
stringency conditions, as defined herein, to all or a portion of a
nucleotide sequence encoding a CD19 binding agent disclosed herein,
or by its complement.
[0205] The present invention encompasses embodiments wherein the
CD19 binding agent is, for example, a humanized full length
antibody, humanized antibody fragment, or a derivative thereof.
[0206] CD19 binding agents can be generated by methods known in the
art. For example, monoclonal antibodies can be prepared using a
wide variety of techniques including, e.g., the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. Hybridoma techniques are generally discussed in, for
example, Harlow et al., Antibodies: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, 2nd ed., 1988; Harlow and Lane,
Using Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, New York (1999); and Hammerling et al., In Monoclonal
Antibodies and T-Cell Hybridomas, pp. 563-681 (Elsevier, N.Y.,
1981). Examples of phage display methods that can be used to make
anti-CD19 antibodies include, e.g., those disclosed in Hoogenboom
and Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J.
Mol. Biol. 222:581; Quan and Carter, 2002, The rise of monoclonal
antibodies as therapeutics in Anti-IgE and Allergic Disease,
Jardieu and Fick Jr., eds., Marcel Dekker, New York, N.Y., Chapter
20, pp. 427-469; Brinkman et al., 1995, J. Immunol. Methods
182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;
Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et
al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in
Immunology 57:191-280; PCT Application No. PCT/GB91/01134; PCT
Publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO
93/11236, WO 95/15982, WO 95/20401, and U.S. Pat. Nos. 5,698,426;
5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743
and 5,969,108 (the disclosures of which are incorporated by
reference herein).
[0207] As discussed herein, the CD19 binding agents can include the
amino acid sequence of a humanized heavy and/or light chain
variable region. Antibodies can be humanized using a variety of
techniques known in the art including, for example, CDR-grafting
(see, e.g., EP 0 239 400; PCT publication WO 91/09967; U.S. Pat.
Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing
(see, e.g., EP 0 592 106; EP 0 519 596; Padlan, Molecular
Immunology, 1991, 28(4/5):489-498; Studnicka et al., 1994, Protein
Engineering 7(6):805-814; Roguska et al., 1994, Proc. Natl. Acad.
Sci. USA 91:969-973), and chain shuffling (see, e.g., U.S. Pat. No.
5,565,332) (all of these references are incorporated by reference
herein). Humanized antibodies and fragments thereof can be produced
by recombinant DNA techniques known in the art, for example using
methods described in International Publication No. WO 87/02671;
European Patent Publication No. 0 184 187; European Patent
Publication No. 0 171 496; European Patent Publication No. 0 173
494; International Publication No. WO 86/01533; U.S. Pat. No.
4,816,567; European Patent Publication No. 0 012 023; Berter et
al., 1988, Science 240:1041-43; Liu et al., 1987, Proc. Natl. Acad.
Sci. USA 84:3439-43; Liu et al., 1987, J. Immunol. 139:3521-26; Sun
et al., 1987, Proc. Natl. Acad. Sci. USA 84:214-18; Nishimura et
al., 1987, Cancer. Res. 47:999-1005; Wood et al., 1985, Nature
314:446-449; Shaw et al., 1988, J. Natl. Cancer Inst. 80:1553-59;
Morrison, 1985, Science 229:1202-07; Oi et al., 1986, BioTechniques
4:214; U.S. Pat. No. 5,225,539; Jones et al., 1986, Nature
321:552-25; Verhoeyan et al., 1988, Science 239:1534; and Beidler
et al., 1988, J. Immunol. 141:4053-60; each of which is
incorporated herein by reference in its entirety.
[0208] Examples of techniques that can be used to produce
single-chain antibodies include those described in U.S. Pat. Nos.
4,946,778 and 5,258,498; Huston et al., 1991, Methods in Enzymology
203:46-88; Shu et al., 1993, Proc. Natl. Acad. Sci. USA
90:7995-7999; and Skerra et al., 1988, Science 240:1038-1040.
[0209] Methods for making bispecific antibodies are known in the
art. Traditional production of full-length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(see, e.g., Milstein et al., 1983, Nature 305:537-39). Because of
the random assortment of immunoglobulin heavy and light chains,
these hybridomas (quadromas) produce a potential mixture of
different antibody molecules, of which one has the correct
bispecific structure. Similar procedures are disclosed in
International Publication No. WO 93/08829, and in Traunecker et
al., 1991, EMBO J. 10:3655-59.
[0210] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences. The
fusion typically is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, C.sub.H2, and
C.sub.H3 regions. In some embodiments, the fusion includes a first
heavy-chain constant region (C.sub.H1) containing the site
necessary for light chain binding, present in at least one of the
fusions. Nucleic acids with sequences encoding the immunoglobulin
heavy chain fusions and, if desired, the immunoglobulin light
chain, are inserted into separate expression vectors, and are
co-transfected into a suitable host organism. This provides for
great flexibility in adjusting the mutual proportions of the three
polypeptide fragments in embodiments when unequal ratios of the
three polypeptide chains used in the construction provide the
optimum yields. It is, however, possible to insert the coding
sequences for two or all three polypeptide chains in one expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios are of no
particular significance.
[0211] In an example of this approach, the bispecific antibodies
have a hybrid immunoglobulin heavy chain with a first binding
specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in
the other arm. This asymmetric structure facilitates the separation
of the desired bispecific compound from unwanted immunoglobulin
chain combinations, as the presence of an immunoglobulin light
chain in only one half of the bispecific molecule provides for a
facile way of separation (see, e.g., International Publication No.
WO 94/04690, which is incorporated herein by reference in its
entirety).
[0212] For further discussion of bispecific antibodies see, for
example, Suresh et al., 1986, Methods in Enzymology 121:210;
Rodrigues et al., 1993, J. Immunology 151:6954-61; Carter et al.,
1992, Bio/Technology 10:163-67; Carter et al., 1995, J.
Hematotherapy 4:463-70; Merchant et al., 1998, Nature Biotechnology
16:677-81. Using such techniques, bispecific antibodies can be
prepared for use in the treatment or prevention of disease as
defined herein.
[0213] Bifunctional antibodies are also described in European
Patent Publication No. 0 105 360. As disclosed in this reference,
hybrid or bifunctional antibodies can be derived biologically,
i.e., by cell fusion techniques, or chemically, especially with
cross-linking agents or disulfide-bridge forming reagents, and may
comprise whole antibodies or fragments thereof. Methods for
obtaining such hybrid antibodies are disclosed for example in
International Publication WO 83/03679 and European Patent
Publication No. 0 217 577, both of which are incorporated herein by
reference.
[0214] A CD19 binding agent can be a derivative of an anti-CD19
antibody. In certain embodiments, an anti-CD19 antibody derivative
comprises an anti-CD19 antibody (e.g., an intact antibody, an
antigen-binding fragment or conservatively substituted polypeptide)
and at least one polypeptide region or other moiety heterologous to
the anti-CD19 antibody. For example, an anti-CD19 antibody can be
modified, e.g., by the covalent attachment of any type of molecule,
such that the covalent attachment does not prevent the antibody
derivative from specifically binding to CD19 via the
antigen-binding region or region derived therefrom, or, if desired,
the effector region or portion thereof from specifically binding Fc
receptor. Typical modifications include, e.g., glycosylation,
deglycosylation, acetylation, pegylation, phosphorylation,
amidation, derivatization by known protecting/blocking groups,
proteolytic cleavage, linkage to a cellular ligand or other
protein, and the like. Any of numerous chemical modifications may
be carried out by known techniques, including, but not limited to
specific chemical cleavage, acetylation, formylation, metabolic
synthesis of tunicamycin, etc.
[0215] In some embodiments, the antibody derivative is a multimer,
such as, for example, a dimer, comprising one or more monomers,
where each monomer includes (i) an antigen-binding region of an
anti-CD19 antibody, or a polypeptide region derived therefrom (such
as, for example, by conservative substitution of one or more amino
acids), and (ii) a multimerizing (e.g., dimerizing) polypeptide
region, such that the antibody derivative forms multimers (e.g.,
homodimers) that specifically bind to CD19. In typical embodiments,
an antigen binding region of an anti-CD19 antibody, or a
polypeptide region derived therefrom, is recombinantly or
chemically fused with a heterologous protein, wherein the
heterologous protein comprises a dimerization or multimerization
domain. Prior to administration of the antibody derivative to a
subject for the purpose of treating or preventing CD19-expressing
cancers, the derivative is subjected to conditions that allow
formation of a homodimer or heterodimer. A heterodimer, as used
herein, may comprise identical dimerization domains but different
CD19 antigen-binding regions, identical CD19 antigen-binding
regions but different dimerization domains, or different CD19
antigen-binding regions and dimerization domains.
[0216] Typical dimerization domains are those that originate from
transcription factors. In one embodiment, the dimerization domain
is that of a basic region leucine zipper ("bZIP") (see, e.g.,
Vinson et al., 1989, Science 246:911-916). Useful leucine zipper
domains include, for example, those of the yeast transcription
factor GCN4, the mammalian transcription factor
CCAAT/enhancer-binding protein C/EBP, and the nuclear transform in
oncogene products, Fos and Jun. (See, e.g., Landschultz et al.,
1988, Science 240:1759-64; Baxevanis and Vinson, 1993, Curr. Op.
Gen. Devel. 3:278-285; O'Shea et al., 1989, Science 243:538-542.)
In another embodiment, the dimerization domain is that of a basic
region helix-loop-helix ("bHLH") protein. (See Murre et al., 1989,
Cell 56:777-783. See also Davis et al., 1990, Cell 60:733-746;
Voronova and Baltimore, 1990, Proc. Natl. Acad. Sci. USA
87:4722-26.) Particularly useful hHLH proteins are myc, max, and
mac.
[0217] In yet other embodiments, the dimerization domain is an
immunoglobulin constant region such as, for example, a heavy chain
constant region or a domain thereof (e.g., a C.sub.H1 domain, a
C.sub.H2 domain, and/or a C.sub.H3 domain). (See, e.g., U.S. Pat.
Nos. 5,155,027; 5,336,603; 5,359,046; and 5,349,053; EP 0 367 166;
and WO 96/04388.)
[0218] Heterodimers are known to form between Fos and Jun (Bohmann
et al., 1987, Science 238:1386-1392), among members of the ATF/CREB
family (Hai et al., 1989, Genes Dev. 3:2083-2090), among members of
the C/EBP family (Cao et al., 1991, Genes Dev. 5:1538-52; Williams
et al., 1991, Genes Dev. 5:1553-67; Roman et al., 1990, Genes Dev.
4:1404-15), and between members of the ATF/CREB and Fos/Jun
families (Hai and Curran, 1991, Proc. Natl. Acad. Sci. USA
88:3720-24). Therefore, when a CD19 binding agent is administered
to a subject as a heterodimer comprising different dimerization
domains, any combination of the foregoing may be used.
[0219] In other embodiments, an anti-CD19 antibody derivative is an
anti-CD19 antibody conjugated to a second antibody (an "antibody
heteroconjugate") (see, e.g. U.S. Pat. No. 4,676,980).
Heteroconjugates can be formed, for example, between an antibody
that binds to CD19 and an antibody that binds to a surface receptor
or receptor complex that mediates ADCC, phagocytosis, and/or CDC,
such as CD16/Fc.gamma.RIII, CD64/Fc.gamma.RI, killer cell
activating or inhibitory receptors, or the complement control
protein CD59. In one embodiment, the binding of the portion of the
multispecific antibody to the second cell surface molecule or
receptor complex enhances the effector functions of an anti-CD19
antibody.
[0220] Antibodies and other binding agents can be assayed for
specific binding to CD19 (e.g., human CD19) by any of various known
methods. Immunoassays which can be used include, for example,
competitive and non-competitive assay systems. Such assays are
routine and well-known in the art. (See, e.g., Ausubel et al.,
eds., Short Protocols in Molecular Biology (John Wiley and Sons,
Inc., New York, 4th ed. 1999); Harlow and Lane, Using Antibodies: A
Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1999.))
[0221] Further, the binding affinity of a CD19 binding agent (e.g.,
anti-CD19 antibody or derivative thereof) to CD19 and the off-rate
of a binding agent-CD19 interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
CD19 (e.g., .sup.3H or .sup.125I) with the antibody of interest in
the presence of increasing amounts of unlabeled CD19, and the
detection of the antibody bound to the labeled CD19. The affinity
of the antibody for CD19 and the binding off-rates can then be
determined from the data by Scatchard plot analysis. Competition
with a second antibody can also be determined using
radioimmunoassays. In this case, CD19 is incubated with the
antibody of interest conjugated to a labeled compound (e.g.,
.sup.3H or .sup.125I) in the presence of increasing amounts of an
unlabeled second antibody. Alternatively, the binding affinity of
an antibody to CD19 and the on- and off-rates of an antibody-CD19
interaction can be determined by surface plasmon resonance. In some
embodiments, the anti-CD19 antibodies or derivatives thereof can be
targeted to and accumulate on the membrane of a CD19-expressing
cell.
[0222] CD19 binding agents (e.g., anti-CD19 antibody or derivative
thereof) can be produced by methods known in the art for the
synthesis of proteins, typically, e.g., by recombinant expression
techniques. Recombinant expression of an antibody or derivative
thereof typically involves construction of an expression vector
containing a nucleic acid that encodes the binding agent. A vector
for the production of the protein molecule may be produced by
recombinant DNA technology using techniques known in the art.
Standard techniques such as, for example, those described in
Sambrook and Russell, Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 3rd ed.,
2001); Sambrook et al., Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2nd
ed., 1989); Short Protocols in Molecular Biology (Ausubel et al.,
John Wiley and Sons, New York, 4th ed., 1999); and Glick and
Pasternak, Molecular Biotechnology: Principles and Applications of
Recombinant DNA (ASM Press, Washington, D.C., 2nd ed., 1998) can be
used for recombinant nucleic acid methods, nucleic acid synthesis,
cell culture, transgene incorporation, and recombinant protein
expression.
[0223] For example, for recombinant expression of an anti-CD19
antibody, an expression vector may encode a heavy or light chain
thereof, or a heavy or light chain variable domain, operably linked
to a promoter. An expression vector may include, for example, the
nucleotide sequence encoding the constant region of the antibody
molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication
WO 89/01036; and U.S. Pat. No. 5,122,464), and the variable domain
of the antibody may be cloned into such a vector for expression of
the entire heavy or light chain. The expression vector is
transferred to a host cell by conventional techniques, and the
transfected cells are then cultured by conventional techniques to
produce the anti-CD19 antibody. In typical embodiments for the
expression of double-chain antibodies, vectors encoding both the
heavy and light chains can be co-expressed in the host cell for
expression of the entire immunoglobulin molecule.
[0224] A variety of prokaryotic and eukaryotic host-expression
vector systems can be utilized to express a CD19 binding agent
(e.g., anti-CD19 antibody or derivative thereof). Typically,
eukaryotic cells, particularly for whole recombinant anti-CD19
antibody molecules, are used for the expression of the recombinant
protein. For example, mammalian cells such as Chinese hamster ovary
cells (CHO; e.g., DG44), in conjunction with a vector such as the
major intermediate early gene promoter element from human
cytomegalovirus, is an effective expression system for the
production of anti-CD19 antibodies and derivatives thereof (see,
e.g., Foecking et al., 1986, Gene 45:101; Cockett et al., 1990,
Bio/Technology 8:2). CD19 binding aagents can also be expressed
using the CHEF system. (See, e.g., U.S. Pat. No. 5,888,809.)
[0225] Other host-expression systems include, for example,
plasmid-based expression systems in bacterial cells (see, e.g.,
Ruther et al., 1983, EMBO 1,2:1791; Inouye and Inouye, 1985,
Nucleic Acids Res. 13:3101-3109; Van Heeke and Schuster, 1989, J.
Biol. Chem. 24:5503-5509); insect systems such as, e.g., the use of
Autographa californica nuclear polyhedrosis virus (AcNPV)
expression vector in Spodoptera frugiperda cells; and viral-based
expression systems in mammalian cells, such as, e.g.,
adenoviral-based systems (see, e.g., Logan and Shenk, 1984, Proc.
Natl. Acad. Sci. USA 81:355-359; Bittner et al., 1987, Methods in
Enzymol. 153:51-544).
[0226] In addition, a host cell strain can be chosen that modulates
the expression of the inserted sequences, or modifies and processes
the gene product in the specific fashion desired. Appropriate cell
lines or host systems can be chosen to ensure the correct
modification and processing (e.g., glycosylation, phosphorylation,
and cleavage) of the protein expressed. To this end, eukaryotic
host cells which possess the cellular machinery for proper
processing of the primary transcript and gene product can be used.
Such mammalian host cells include, for example, CHO, VERO, BHK,
HeLa, COS, MDCK, 293, 3T3, and W138.
[0227] A stable expression system is typically used for long-term,
high-yield production of a recombinant CD19 binding agent. For
example, cell lines that stably express the anti-CD19 antibody or
derivative thereof can be engineered by transformation of host
cells with DNA controlled by appropriate expression control
elements (e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation sites) and a selectable marker,
followed by growth of the transformed cells in a selective media.
The selectable marker confers resistance to the selection and
allows cells to stably integrate the DNA into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. A number of selection systems can be used, including,
for example, the herpes simplex virus thymidine kinase,
hypoxanthineguanine phosphoribosyltransferase, and adenine
phosphoribosyltransferase genes, which can be employed in tk.sup.-,
hgprt.sup.- or aprt.sup.- cells, respectively. Also, antimetabolite
resistance can be used as the basis of selection for the following
genes: dhfr, which confers resistance to methotrexate; gpt, which
confers resistance to mycophenolic acid; neo, which confers
resistance to the aminoglycoside G-418; and hygro, which confers
resistance to hygromycin. Methods commonly known in the art of
recombinant DNA technology can be routinely applied to select the
desired recombinant clone, and such methods are described, for
example, in Ausubel et al., eds., in the Current Protocols in
Molecular Biology series of laboratory technique manuals, 1987-1999
Current Protocols, .COPYRGT. 1994-1999 John Wiley and Sons, Inc.).;
Kriegler, Gene Transfer and Expression, A Laboratory Manual
(Stockton Press, N.Y., 1990); Current Protocols in Human Genetics
(Dracopoli et al. eds., John Wiley and Sons, N.Y., 1994, Chapters
12 and 13); and Colberre-Garapin et al., 1981, J. Mol. Biol.
150:1.
[0228] The expression levels of an antibody or derivative can be
increased by vector amplification. (See generally Bebbington and
Hentschel, The Use of Vectors Based on Gene Amplification for the
Expression of Cloned Genes in Mammalian Cells in DNA Cloning, Vol.
3 (Academic Press, New York, 1987).) When a marker in the vector
system expressing an anti-CD19 antibody or derivative thereof is
amplifiable, an increase in the level of inhibitor present in host
cell culture media will select host cells that have increased copy
number of a marker gene conferring resistance to the inhibitor. The
copy number of an associated antibody gene will also be increased,
thereby increasing expression of the antibody or derivative thereof
(see, e.g., Crouse et al., 1983, Mol. Cell. Biol. 3:257).
[0229] Where a CD19 binding agent comprises both a heavy and a
light chain, the host cell may be co-transfected with two
expression vectors, the first vector encoding the heavy chain
protein and the second vector encoding the light chain protein. The
two vectors may contain identical selectable markers which enable
equal expression of heavy and light chain proteins. Alternatively,
a single vector may be used which encodes, and is capable of
expressing, both heavy and light chain proteins. In such
situations, the light chain is typically placed before the heavy
chain to avoid an excess of toxic free heavy chain (see, e.g.,
Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad.
Sci. USA 77:2197). The coding sequences for the heavy and light
chains may comprise cDNA or genomic DNA.
[0230] Once a CD19 binding agent has been produced (e.g., by an
animal, chemical synthesis, or recombinant expression), it can be
purified by any suitable method for purification of proteins,
including, for example, by chromatography (e.g., ion exchange or
affinity chromatography (such as, for example, Protein A
chromatography for purification of antibodies having an intact Fc
region)), centrifugation, differential solubility, or by any other
standard technique for the purification of proteins. An anti-CD19
antibody or derivative thereof can, for example, be fused to a
marker sequence, such as a peptide, to facilitate purification by
affinity chromatography. Suitable marker amino acid sequences
include, e.g., a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., Chatsworth, Calif., 91311), and the
"HA" tag, which corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767),
and the "flag" tag.
[0231] Typically, the CD19 binding agent is substantially purified
(e.g., substantially free from substances that limit its effect or
produce undesired side-effects). In some embodiments, the CD19
binding agent is at least about 40% pure, at least about 50% pure,
or at least about 60% pure. In some embodiments, the CD19 binding
agent is at least about 60-65%, 65-70%, 70-75%, 75-80%, 80-85%,
85-90%, 90-95%, or 95-98% pure. In some embodiments, the CD19
binding agent is approximately 99% pure.
[0232] Further CD19 binding agents can include fusion proteins
(i.e., proteins that are recombinantly fused or chemically
conjugated, including both covalent and non-covalent conjugation)
to heterologous proteins (of typically at least 10, 20, 30, 40, 50,
60, 70, 80, 90 or at least 100 amino acids). In some embodiments,
such a CD19 binding agent includes the amino acid sequence of a
humanized heavy and/or light chain variable region that
specifically binds to CD19 and optionally an immunoglobulin
effector region or a functional equivalent thereof. As used herein,
a functional equivalent of an immunoglobulin effector region binds
to an Fc receptor on an immune cell with phagocytic or lytic
activity, or the immunoglobulin effector region binds to one or
more components of the complement system. The linkage of the CD19
binding portion to the heterologous protein is not necessarily
direct, but may occur through a linker sequence(s).
[0233] For example, a CD19 binding agent can be produced
recombinantly by fusing a humanized variable region in frame with a
sequence coding for a heterologous protein. The heterologous
protein optionally can include an effector region or a functional
equivalent thereof and may provide one or more of the following
characteristics: promote stable expression; provide a means of
facilitating high yield recombinant expression; and/or provide a
multimerization domain.
[0234] A CD19 binding agent can be identified using any method
suitable for screening for protein-protein interactions. Typically,
proteins are initially identified by their ability to specifically
bind to CD19. Among the traditional methods which can be employed
are "interaction cloning" techniques which entail probing
expression libraries with labeled CD19 in a manner similar to the
technique of antibody probing of .lamda.gt11 libraries. By way of
example and not limitation, this can be achieved as follows: a cDNA
clone encoding CD19 can be modified at the C-terminus by inserting
the phosphorylation site for the heart muscle kinase (HMK) (see,
e.g., Blanar and Rutter, 1992, Science 256:1014-18). The
recombinant protein is expressed in E. coli and purified on a
GDP-affinity column to homogeneity (Edery et al., 1988, Gene
74:517-25) and labeled using .gamma..sup.32P-ATP and bovine heart
muscle kinase (Sigma-Aldrich Co., St. Louis, Mo.) to a specific
activity of 1.times.10.sup.8 cpm/.mu.g, and used to screen a human
placenta .lamda.gt11 cDNA library in a "far-Western assay" (Blanar
and Rutter, 1992, Science 256:1014-18). Plaques that interact with
the CD19 probe are isolated. The cDNA inserts of positive .lamda.
plaques are released and subcloned into a vector suitable for
sequencing, such as pBluescript KS (Stratagene, La Jolla,
Calif.).
[0235] One method which detects protein interactions in vivo is the
two-hybrid system. One version of this system has been described
(Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88:9578-82) and is
commercially available from Clontech (Palo Alto, Calif.).
[0236] Anti-CD19 ligand-drug conjugate compounds can include useful
classes of cytotoxic or immunomodulatory agents, for example,
antitubulin agents, auristatins, DNA minor groove binders, DNA
replication inhibitors, alkylating agents (e.g., platinum complexes
such as cis-platin, mono(platinum), bis(platinum) and tri-nuclear
platinum complexes and carboplatin), anthracyclines, antibiotics,
antifolates, antimetabolites, chemotherapy sensitizers,
duocarmycins, etoposides, fluorinated pyrimidines, ionophores,
lexitropsins, nitrosoureas, platinols, pre-forming compounds,
purine antimetabolites, puromycins, radiation sensitizers,
steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or
the like.
[0237] Chemotherapeutic agents can be used in a ligand-drug
conjugate, or in combination with anti-CD19 antibodies or anti-CD19
ligand-drug conjugate, in methods for treatment of neoplastic
disease. In certain embodiments, an antibody-cytotoxin conjugate
comprising antibodies to CD19 can be used to boost immunity induced
through standard cancer treatments. In these instances, it can be
possible to reduce the dose of chemotherapeutic reagent
administered (Mokyr et al., Cancer Research 58: 5301-5304, 1998).
The scientific rationale behind the combined use of CD19 antibody
and chemotherapy is that cell death, that is a consequence of the
cytotoxic action of most chemotherapeutic compounds, should result
in increased levels of tumor antigen in the antigen presentation
pathway. Thus, CD19 antibody can boost an immune response primed to
chemotherapy release of tumor cells.
[0238] B cell lymphoma and leukemia, can include, but are not
limited to, non-Hodgkin lymphoma, chronic lymphocytic leukemia, and
acute lymphoblastic leukemia
[0239] "Leukemia" refers to progressive, malignant diseases of the
blood-forming organs and is generally characterized by a distorted
proliferation and development of leukocytes and their precursors in
the blood and bone marrow. Leukemia is generally clinically
classified on the basis of (1) the duration and character of the
disease--acute or chronic; (2) the type of cell involved; myeloid
(myelogenous), lymphoid (lymphogenous), or monocytic; and (3) the
increase or non-increase in the number of abnormal cells in the
blood--leukemic or aleukemic (subleukemic). Leukemia includes, for
example, acute nonlymphocytic leukemia, chronic lymphocytic
leukemia, acute granulocytic leukemia, chronic granulocytic
leukemia, acute promyelocytic leukemia, adult T-cell leukemia,
aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia,
blast cell leukemia, bovine leukemia, chronic myelocytic leukemia,
leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross'
leukemia, hairy-cell leukemia, hemoblastic leukemia,
hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia,
acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia,
lymphoblastic leukemia, lymphocytic leukemia, lymphogenous
leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell
leukemia, megakaryocytic leukemia, micromyeloblastic leukemia,
monocytic leukemia, myeloblastic leukemia, myelocytic leukemia,
myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli
leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic
leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell
leukemia, subleukemic leukemia, and undifferentiated cell
leukemia.
Ligand-Drug Conjugate Compounds
[0240] The present invention provides, inter alia, ligand-drug
conjugate compounds for targeted delivery of drugs. The inventors
have made the discovery that the ligand-drug conjugate compounds
have potent cytotoxic and/or cytostatic activity against B cells
expressing CD19. The ligand-drug conjugate compounds comprise a
Ligand unit covalently linked to at least one Drug unit. The Drug
units can be covalently linked directly or via a Linker unit
(-LU--).
[0241] In some embodiments, the ligand drug conjugate compound has
the following formula:
L-(LU-D).sub.p (I)
[0242] or a pharmaceutically acceptable salt or solvate thereof;
wherein:
[0243] L is the Ligand unit, i.e., CD19 binding agent of the
present invention, and
[0244] (LU-D) is a Linker unit-Drug unit moiety, wherein:
[0245] LU- is a Linker unit, and
[0246] -D is a drug unit having cytostatic or cytotoxic activity
against a target cell; and
[0247] p is an integer from 1 to about 20.
[0248] In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8,
1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some
embodiments, p ranges from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6,
2 to 5, 2 to 4 or 2 to 3. In other embodiments, p is 1, 2, 3, 4, 5
or 6. In some embodiments, p is 2 or 4.
[0249] In some embodiments, the ligand drug conjugate compound has
the following formula:
L-(A.sub.a-W.sub.w--Y.sub.y-D).sub.p (II)
[0250] or a pharmaceutically acceptable salt or solvate
thereof;
[0251] wherein:
[0252] L is the Ligand unit, i.e. CD19 binding agent; and
[0253] -A.sub.a-W.sub.w--Y.sub.y-- is a Linker unit (LU),
wherein:
[0254] -A- is a Stretcher unit,
[0255] a is 0 or 1,
[0256] each --W-- is independently an Amino Acid unit,
[0257] w is an integer ranging from 0 to 12,
[0258] --Y-- is a self-immolative spacer unit,
[0259] y is 0, 1 or 2;
[0260] -D is a drug units having cytostatic or cytotoxic activity
against the target cell; and
[0261] p is an integer from 1 to about 20.
[0262] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1
or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or
1. In some embodiments, p ranges from 1 to 10, 1 to 9, 1 to 8, 1 to
7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments,
p ranges from 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4 or 2 to 3. In
other embodiments, p is 1, 2, 3, 4, 5 or 6. In some embodiments, p
is 2 or 4. In some embodiments, when w is not zero, y is 1 or
2.
[0263] The drug loading is represented by p, the average number of
drug molecules per Ligand, e.g., antibody in a molecule. Drug
loading may range from 1 to 20 drugs (D) per Ligand. The average
number of drugs per ligand in preparation of conjugation reactions
may be characterized by conventional means such as mass
spectroscopy, ELISA assay, and HPLC. The quantitative distribution
of Ligand-Drug-Conjugates in terms of p may also be determined. In
some instances, separation, purification, and characterization of
homogeneous Ligand-Drug-conjugates where p is a certain value from
Ligand-Drug-Conjugates with other drug loadings may be achieved by
means such as reverse phase HPLC or electrophoresis. In exemplary
embodiments, p is from 2 to 8.
[0264] The generation of ligand-drug conjugate compounds can be
accomplished by any technique known to the skilled artisan.
Briefly, the ligand-drug conjugate compounds comprise a CD19
binding agent as the ligand unit, a drug, and optionally a linker
that joins the drug and the binding agent. A number of different
reactions are available for covalent attachment of drugs and/or
linkers to binding agents. This is often accomplished by reaction
of the amino acid residues of the binding agent, e.g., antibody
molecule, including the amine groups of lysine, the free carboxylic
acid groups of glutamic and aspartic acid, the sulfhydryl groups of
cysteine and the various moieties of the aromatic amino acids. One
of the most commonly used non-specific methods of covalent
attachment is the carbodiimide reaction to link a carboxy (or
amino) group of a compound to amino (or carboxy) groups of the
antibody. Additionally, bifunctional agents such as dialdehydes or
imidoesters have been used to link the amino group of a compound to
amino groups of an antibody molecule. Also available for attachment
of drugs to binding agents is the Schiff base reaction. This method
involves the periodate oxidation of a drug that contains glycol or
hydroxy groups, thus forming an aldehyde which is then reacted with
the binding agent. Attachment occurs via formation of a Schiff base
with amino groups of the binding agent. Isothiocyanates can also be
used as coupling agents for covalently attaching drugs to binding
agents. Other techniques are known to the skilled artisan and
within the scope of the present invention.
[0265] In certain embodiments, an intermediate, which is the
precursor of the linker, is reacted with the drug under appropriate
conditions. In certain embodiments, reactive groups are used on the
drug and/or the intermediate. The product of the reaction between
the drug and the intermediate, or the derivatized drug, is
subsequently reacted with the CD19 binding agent under appropriate
conditions.
[0266] Each of the particular units of the ligand-drug conjugate
compounds is described in more detail herein. The synthesis and
structure of linker units, stretcher units, amino acid units,
self-immolative spacer unit, and drug units are also described in
U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649
and 2005-0009751, each if which is incorporated herein by reference
in its entirety and for all purposes.
Linker Units
[0267] Typically, the ligand-drug conjugate compounds comprise a
linker region between the drug unit and the ligand unit. In some
embodiments, the linker is cleavable under intracellular
conditions, such that cleavage of the linker releases the drug unit
from the ligand in the intracellular environment.
[0268] For example, in some embodiments, the linker is cleavable by
a cleaving agent that is present in the intracellular environment
(e.g., within a lysosome or endosome or caveolea). The linker can
be, e.g., a peptidyl linker that is cleaved by an intracellular
peptidase or protease enzyme, including, but not limited to, a
lysosomal or endosomal protease. Typically, the peptidyl linker is
at least two amino acids long or at least three amino acids long.
Cleaving agents can include cathepsins B and D and plasmin, all of
which are known to hydrolyze dipeptide drug derivatives resulting
in the release of active drug inside target cells (see, e.g.,
Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most
typical are peptidyl linkers that are cleavable by enzymes that are
present in CD19-expressing cells. For example, a peptidyl linker
that is cleavable by the thiol-dependent protease cathepsin-B,
which is highly expressed in cancerous tissue, can be used (e.g., a
Phe-Leu or a Gly-Phe-Leu-Gly linker). Other such linkers are
described, e.g., in U.S. Pat. No. 6,214,345, incorporated herein by
reference in its entirety and for all purposes. In a specific
embodiment, the peptidyl linker cleavable by an intracellular
protease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S.
Pat. No. 6,214,345, which describes the synthesis of doxorubicin
with the val-cit linker). One advantage of using intracellular
proteolytic release of the therapeutic agent is that the agent is
typically attenuated when conjugated and the serum stabilities of
the conjugates are typically high.
[0269] In other embodiments, the cleavable linker is pH-sensitive,
i.e., sensitive to hydrolysis at certain pH values. Typically, the
pH-sensitive linker hydrolyzable under acidic conditions. For
example, an acid-labile linker that is hydrolyzable in the lysosome
(e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic
amide, orthoester, acetal, ketal, or the like) can be used. (See,
e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and
Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989,
Biol. Chem. 264:14653-14661.) Such linkers are relatively stable
under neutral pH conditions, such as those in the blood, but are
unstable at below pH 5.5 or 5.0, the approximate pH of the
lysosome. In certain embodiments, the hydrolyzable linker is a
thioether linker (such as, e.g., a thioether attached to the
therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat.
No. 5,622,929).
[0270] In yet other embodiments, the linker is cleavable under
reducing conditions (e.g., a disulfide linker). A variety of
disulfide linkers are known in the art, including, for example,
those that can be formed using SATA
(N-succinimidyl-S-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
, SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res.
47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody
Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,
Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
[0271] In yet other specific embodiments, the linker is a malonate
linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a
maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem.
3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995,
Bioorg-Med-Chem. 3(10):1305-12).
[0272] Typically, the linker is not substantially sensitive to the
extracellular environment. As used herein, "not substantially
sensitive to the extracellular environment," in the context of a
linker, means that no more than about 20%, typically no more than
about 15%, more typically no more than about 10%, and even more
typically no more than about 5%, no more than about 3%, or no more
than about 1% of the linkers, in a sample of ligand-drug conjugate
compound, are cleaved when the ligand-drug conjugate compound
presents in an extracellular environment (e.g., in plasma). Whether
a linker is not substantially sensitive to the extracellular
environment can be determined, for example, by incubating with
plasma the ligand-drug conjugate compound for a predetermined time
period (e.g., 2, 4, 8, 16, or 24 hours) and then quantitating the
amount of free drug present in the plasma.
[0273] In other, non-mutually exclusive embodiments, the linker
promotes cellular internalization. In certain embodiments, the
linker promotes cellular internalization when conjugated to the
therapeutic agent (i.e., in the milieu of the linker-therapeutic
agent moiety of the ligand-drug conjugate compound as described
herein). In yet other embodiments, the linker promotes cellular
internalization when conjugated to both the auristatin compound and
the anti-CD19 antibody.
[0274] A variety of linkers that can be used with the present
compositions and methods are described in WO 2004010957 entitled
"Drug Conjugates and Their Use for Treating Cancer, An Autoimmune
Disease or an Infectious Disease" filed Jul. 31, 2003, and U.S.
Publication No. 2006/0074008 entitled "Drug Conjugates and their
use for treating cancer, an autoimmune disease or an infectious
disease", filed Jul. 31, 2002 (the disclosure of which is
incorporated by reference herein in its entirety and for all
purposes).
[0275] A "Linker unit" (LU) is a bifunctional compound that can be
used to link a Drug unit and a Ligand unit to form a ligand-drug
conjugate compound. In some embodiments, the Linker unit has the
formula:
-A.sub.a-W.sub.w--Y.sub.y--
[0276] wherein:-A- is a Stretcher unit,
[0277] a is 0 or 1,
[0278] each --W-- is independently an Amino Acid unit,
[0279] w is an integer ranging from 0 to 12,
[0280] --Y-- is a self-immolative Spacer unit, and
[0281] y is 0, 1 or 2.
[0282] In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0, 1
or 2. In some embodiments, a is 0 or 1, w is 0 or 1, and y is 0 or
1.
The Stretcher Unit
[0283] The Stretcher unit (A), when present, is capable of linking
a Ligand unit to an Amino Acid unit (--W--), if present, to a
Spacer unit (--Y--), if present; or to a Drug unit (-D). Useful
functional groups that can be present on a CD19 binding agent,
either naturally or via chemical manipulation include, but are not
limited to, sulfhydryl, amino, hydroxyl, the anomeric hydroxyl
group of a carbohydrate, and carboxyl. Suitable functional groups
are sulfhydryl and amino. In one example, sulfhydryl groups can be
generated by reduction of the intramolecular disulfide bonds of an
anti-CD19 antibody. In another embodiment, sulfhydryl groups can be
generated by reaction of an amino group of a lysine moiety of an
anti-CD19 antibody with 2-iminothiolane (Traut's reagent) or other
sulfhydryl generating reagents. In certain embodiments, the
anti-CD19 antibody is a recombinant antibody and is engineered to
carry one or more lysines. In certain other embodiments, the
recombinant anti-CD19 antibody is engineered to carry additional
sulfhydryl groups, e.g., additional cysteines.
[0284] In one embodiment, the Stretcher unit forms a bond with a
sulfur atom of the Ligand unit. The sulfur atom can be derived from
a sulfhydryl group of a Ligand. Representative Stretcher units of
this embodiment are depicted within the square brackets of Formulas
IIIa and IIIb, wherein L-, --W--, --Y--, -D, w and y are as defined
above, and R.sub.17 is selected from --C.sub.1-C.sub.10 alkylene-,
--C.sub.1-C.sub.10 alkenylene-, --C.sub.1-C.sub.10 alkynylene-,
carbocyclo-, --O--(C.sub.1-C.sub.8 alkylene)-, O--(C.sub.1-C.sub.8
alkenylene)-, --O--(C.sub.1-C.sub.8 alkynylene)-, -arylene-,
alkylene-arylene-, --C.sub.2-C.sub.10 alkenylene-arylene,
--C.sub.2-C.sub.10 alkynylene-arylene, -arylene-C.sub.1-C.sub.10
alkylene-, -arylene-C.sub.2-C.sub.10 alkenylene-,
-arylene-C.sub.2-C.sub.10 alkynylene-, --C.sub.1-C.sub.10
alkylene-(carbocyclo)-, --C.sub.2-C.sub.10
alkenylene-(carbocyclo)-, --C.sub.2-C.sub.10
alkynylene-(carbocyclo)-, -(carbocyclo)-C.sub.1-C.sub.10 alkylene-,
-(carbocyclo)-C.sub.2-C.sub.10 alkenylene-,
-(carbocyclo)-C.sub.2-C.sub.10 alkynylene, -heterocyclo-,
alkylene-(heterocyclo)-, --C.sub.2-C.sub.10
alkenylene-(heterocyclo)-, --C.sub.2-C.sub.10
alkynylene-(heterocyclo)-, -(heterocyclo)-C.sub.1-C.sub.10
alkylene-, -(heterocyclo)-C.sub.2-C.sub.10 alkenylene-,
-(heterocyclo)-C.sub.1-C.sub.10 alkynylene-,
--(CH.sub.2CH.sub.2O).sub.r--, or
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--, and r is an integer
ranging from 1-10, wherein said alkyl, alkenyl, alkynyl, alkylene,
alkenylene, alkynyklene, aryl, carbocyle, carbocyclo, heterocyclo,
and arylene radicals, whether alone or as part of another group,
are optionally substituted. Alkylene, alkenylene, alkynylene
radicals, whether alone or as part of another group, can be
optionally substituted with, for example, one or more groups
independently selected from A1; carbocyclo radicals, whether alone
or as part of another group, can be optionally substituted with,
for example, one or more groups independently selected from A2;
arylene radicals, whether alone or as part of another group, can be
optionally substituted with, for example, one or more groups
independently selected from A3; heterocyclo radicals, whether alone
or as part of another group, can be optionally substituted with,
for example, one or more groups independently selected from A4. A1,
A2, A3, and A4 are as defined herein. It is to be understood from
all the exemplary embodiments that even where not denoted
expressly, from 1 to 20 drug moieties can be linked to a Ligand
(p=1-20).
##STR00002##
[0285] An illustrative Stretcher unit is that of Formula Ma wherein
R.sup.17 is --(CH.sub.2).sub.5--:
##STR00003##
[0286] Another illustrative Stretcher unit is that of Formula IIIa
wherein R.sup.17 is --(CH.sub.2CH.sub.2O).sub.r--CH.sub.2--; and r
is 2:
##STR00004##
[0287] Still another illustrative Stretcher unit is that of Formula
Mb wherein R.sup.17 is --(CH.sub.2).sub.5--:
##STR00005##
[0288] In certain embodiments, the Stretcher unit is linked to the
Ligand unit via a disulfide bond between a sulfur atom of the
Ligand unit and a sulfur atom of the Stretcher unit. A
representative Stretcher unit of this embodiment is depicted within
the square brackets of Formula IV, wherein R.sup.17, L-, --W--,
--Y--, -D, w and y are as defined above.
L-S S--R.sup.17--C(O) W.sub.w--Y.sub.y-D IV
[0289] It should be noted that throughout this application, the S
moiety in the formula below refers to a sulfur atom of the Ligand
unit, unless otherwise indicated by context.
##STR00006##
[0290] In yet other embodiments, the Stretcher contains a reactive
site that can form a bond with a primary or secondary amino group
of a Ligand. Examples of these reactive sites include, but are not
limited to, activated esters such as succinimide esters, 4
nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl
esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates
and isothiocyanates. Representative Stretcher units of this
embodiment are depicted within the square brackets of Formulas Va
and Vb, wherein --R.sup.17--, L-, --W--, --Y--, -D, w and y are as
defined above;
##STR00007##
[0291] In some embodiments, the Stretcher contains a reactive site
that is reactive to a modified carbohydrate's (--CHO) group that
can be present on a Ligand. For example, a carbohydrate can be
mildly oxidized using a reagent such as sodium periodate and the
resulting (--CHO) unit of the oxidized carbohydrate can be
condensed with a Stretcher that contains a functionality such as a
hydrazide, an oxime, a primary or secondary amine, a hydrazine, a
thiosemicarbazone, a hydrazine carboxylate, and an arylhydrazide
such as those described by Kaneko et al., 1991, Bioconjugate Chem.
2:133-41. Representative Stretcher units of this embodiment are
depicted within the square brackets of Formulas VIa, VIb, and VIc,
wherein --R.sup.17--, L-, --W--, --Y--, -D, w and y are as defined
as above.
##STR00008##
The Amino Acid Unit
[0292] The Amino Acid unit (--W--), when present, links the
Stretcher unit to the Spacer unit if the Spacer unit is present,
links the Stretcher unit to the Drug moiety if the Spacer unit is
absent, and links the Ligand unit to the Drug unit if the Stretcher
unit and Spacer unit are absent.
[0293] W.sub.w-- can be, for example, a dipeptide, tripeptide,
tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide,
nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Each
--W-- unit independently has the formula denoted below in the
square brackets, and w is an integer ranging from 0 to 12:
##STR00009##
[0294] wherein R.sup.19 is hydrogen, methyl, isopropyl, isobutyl,
sec-butyl, benzyl, p-hydroxybenzyl, --CH.sub.2OH, --CH(OH)CH.sub.3,
--CH.sub.2CH.sub.2SCH.sub.3, --CH.sub.2CONH.sub.2, --CH.sub.2COOH,
--CH.sub.2CH.sub.2CONH.sub.2, --CH.sub.2CH.sub.2COOH,
--(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.3NH.sub.2,
--(CH.sub.2).sub.3NHCOCH.sub.3, --(CH.sub.2).sub.3NHCHO,
--(CH.sub.2).sub.4NHC(.dbd.NH)NH.sub.2, --(CH.sub.2).sub.4NH.sub.2,
--(CH.sub.2).sub.4NHCOCH.sub.3, --(CH.sub.2).sub.4NHCHO,
--(CH.sub.2).sub.3NHCONH.sub.2, --(CH.sub.2).sub.4NHCONH.sub.2,
--CH.sub.2CH.sub.2CH(OH)CH.sub.2NH.sub.2, 2-pyridylmethyl-,
3-pyridylmethyl-, 4-pyridylmethyl-, phenyl, cyclohexyl,
##STR00010##
[0295] In some embodiments, the Amino Acid unit can be
enzymatically cleaved by one or more enzymes, including a cancer or
tumor-associated protease, to liberate the Drug unit (-D), which in
one embodiment is protonated in vivo upon release to provide a Drug
(D).
[0296] In certain embodiments, the Amino Acid unit can comprise
natural amino acids. In other embodiments, the Amino Acid unit can
comprise non-natural amino acids. Illustrative Ww units are
represented by formulas (VII)-(IX):
##STR00011##
wherein R.sup.20 and R.sup.21 are as follows:
TABLE-US-00002 (VIII) ##STR00012## R.sup.20 R.sup.21 Benzyl
(CH.sub.2).sub.4NH.sub.2; methyl (CH.sub.2).sub.4NH.sub.2;
isopropyl (CH.sub.2).sub.4NH.sub.2; isopropyl
(CH.sub.2).sub.3NHCONH.sub.2; benzyl (CH.sub.2).sub.3NHCONH.sub.2;
isobutyl (CH.sub.2).sub.3NHCONH.sub.2; sec-butyl
(CH.sub.2).sub.3NHCONH.sub.2; ##STR00013##
(CH.sub.2).sub.3NHCONH.sub.2; benzyl methyl; benzyl
(CH.sub.2).sub.3NHC(.dbd.NH)NH.sub.2;
wherein R.sup.20, R.sup.21 and R.sup.22 are as follows:
TABLE-US-00003 (IX) ##STR00014## R.sup.20 R.sup.21 R.sup.22 benzyl
benzyl (CH.sub.2).sub.4NH.sub.2; isopropyl benzyl
(CH.sub.2).sub.4NH.sub.2; and H benzyl
(CH.sub.2).sub.4NH.sub.2;
wherein R.sup.20, R.sup.21, R.sup.22 and R.sup.23 are as
follows:
TABLE-US-00004 R.sup.20 R.sup.21 R.sup.22 R.sup.23 H benzyl
isobutyl H; and methyl isobutyl methyl isobutyl.
[0297] Exemplary Amino Acid units include, but are not limited to,
units of formula VII where: R.sup.20 is benzyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; R.sup.20 is isopropyl and R.sup.21 is
--(CH.sub.2).sub.4NH.sub.2; or R.sup.20 is isopropyl and R.sup.21
is --(CH.sub.2).sub.3NHCONH.sub.2. Another exemplary Amino Acid
unit is a unit of formula VII wherein R.sup.20 is benzyl, R.sup.21
is benzyl, and R.sup.22 is --(CH.sub.2).sub.4NH.sub.2.
[0298] Useful --W.sub.w-- units can be designed and optimized in
their selectivity for enzymatic cleavage by a particular enzyme,
for example, a tumor-associated protease. In one embodiment, a
--W.sub.w-- unit is that whose cleavage is catalyzed by cathepsin
B, C and D, or a plasmin protease.
[0299] In one embodiment, --W.sub.w-- is a dipeptide, tripeptide,
tetrapeptide or pentapeptide. When R.sup.19, R.sup.20, R.sup.21,
R.sup.22 or R.sup.23 is other than hydrogen, the carbon atom to
which R.sup.19, R.sup.20, R.sup.21, R.sup.22 or R.sup.23 is
attached is chiral.
[0300] Each carbon atom to which R.sup.19, R.sup.20, R.sup.21,
R.sup.22 or R.sup.23 is attached is independently in the (S) or (R)
configuration.
[0301] In one aspect of the Amino Acid unit, the Amino Acid unit is
valine-citrulline (vc or val-cit). In another aspect, the Amino
Acid unit is phenylalanine-lysine (i.e., fk). In yet another aspect
of the Amino Acid unit, the Amino Acid unit is
N-methylvaline-citrulline. In yet another aspect, the Amino Acid
unit is 5-aminovaleric acid, homo phenylalanine lysine,
tetraisoquinolinecarboxylate lysine, cyclohexylalanine lysine,
isonepecotic acid lysine, beta-alanine lysine, glycine serine
valine glutamine and isonepecotic acid.
The Spacer Unit
[0302] The Spacer unit (--Y--), when present, links an Amino Acid
unit to the Drug unit when an Amino Acid unit is present.
Alternately, the Spacer unit links the Stretcher unit to the Drug
unit when the Amino Acid unit is absent. The Spacer unit also links
the Drug unit to the Ligand unit when both the Amino Acid unit and
Stretcher unit are absent.
[0303] Spacer units are of two general types: non self-immolative
or self-immolative. A non self-immolative Spacer unit is one in
which part or all of the Spacer unit remains bound to the Drug
moiety after cleavage, particularly enzymatic, of an Amino Acid
unit from the ligand-drug conjugate. Examples of a non
self-immolative Spacer unit include, but are not limited to a
(glycine-glycine) Spacer unit and a glycine Spacer unit (both
depicted in Scheme 1) (infra). When a conjugate containing a
glycine-glycine Spacer unit or a glycine Spacer unit undergoes
enzymatic cleavage via an enzyme (e.g., a tumor-cell
associated-protease, a cancer-cell-associated protease or a
lymphocyte-associated protease), a glycine-glycine-Drug moiety or a
glycine-Drug moiety is cleaved from L-Aa-Ww-. In one embodiment, an
independent hydrolysis reaction takes place within the target cell,
cleaving the glycine-Drug moiety bond and liberating the Drug.
##STR00015##
[0304] In some embodiments, a non self-immolative Spacer unit
(--Y--) is -Gly-. In some embodiments, a non self-immolative Spacer
unit (--Y--) is -Gly-Gly-.
[0305] In one embodiment, a Drug-Linker conjugate is provided in
which the Spacer unit is absent (y=0), or a pharmaceutically
acceptable salt or solvate thereof.
[0306] Alternatively, a conjugate containing a self-immolative
Spacer unit can release -D. As used herein, the term
"self-immolative Spacer" refers to a bifunctional chemical moiety
that is capable of covalently linking together two spaced chemical
moieties into a stable tripartite molecule. It will spontaneously
separate from the second chemical moiety if its bond to the first
moiety is cleaved.
[0307] In some embodiments, --Y.sub.y-- is a p-aminobenzyl alcohol
(PAB) unit (see Schemes 2 and 3) whose phenylene portion is
substituted with Q.sub.m wherein Q is --C.sub.1-C.sub.8 alkyl,
--C.sub.1-C.sub.8 alkenyl, --C.sub.1-C.sub.8 alkynyl,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.1-C.sub.8 alkenyl),
--O--(C.sub.1-C.sub.8 alkynyl), -halogen,-nitro or -cyano; and m is
an integer ranging from 0-4. The akyl, alkenyl and alkynyl groups,
whether alone or as part of another group, can be optionally
substituted with R1 as defined herein.
[0308] In some embodiments, --Y-- is a PAB group that is linked to
--W.sub.w-- via the amino nitrogen atom of the PAB group, and
connected directly to -D via a carbonate, carbamate or ether group.
Without being bound by any particular theory or mechanism, Scheme 2
depicts a possible mechanism of Drug release of a PAB group which
is attached directly to -D via a carbamate or carbonate group as
described by Toki et al., 2002, J. Org. Chem. 67:1866-1872.
##STR00016##
[0309] In Scheme 2, Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p
ranges from 1 to about 20. The akyl, alkenyl and alkynyl groups,
whether alone or as part of another group, can be optionally
substituted with A1 as defined herein.
[0310] Without being bound by any particular theory or mechanism,
Scheme 3 depicts a possible mechanism of Drug release of a PAB
group which is attached directly to -D via an ether or amine
linkage, wherein D includes the oxygen or nitrogen group that is
part of the Drug unit.
##STR00017##
[0311] In Scheme 3, Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; and p
ranges from 1 to about 20. The akyl, alkenyl and alkynyl groups,
whether alone or as part of another group, can be optionally
substituted with A1 as defined herein.
[0312] Other examples of self-immolative spacers include, but are
not limited to, aromatic compounds that are electronically similar
to the PAB group such as 2-aminoimidazol-5-methanol derivatives
(Hay et al., 1999, Bioorg. Med. Chem. Lett. 9:2237) and ortho or
para-aminobenzylacetals. Spacers can be used that undergo
cyclization upon amide bond hydrolysis, such as substituted and
unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995,
Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1]
and bicyclo[2.2.2] ring systems (Storm et al., 1972, J. Amer. Chem.
Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry et
al., 1990, J. Org. Chem. 55:5867). Elimination of amine-containing
drugs that are substituted at the .alpha.-position of glycine
(Kingsbury et al., 1984, J. Med. Chem. 27:1447) are also examples
of self-immolative spacers.
[0313] In one embodiment, the Spacer unit is a branched
bis(hydroxymethyl)-styrene (BHMS) unit as depicted in Scheme 4,
which can be used to incorporate and release multiple drugs.
##STR00018##
[0314] In Scheme 4, Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; m is an integer ranging from 0-4; n is
0 or 1; and p ranges raging from 1 to about 20. The akyl, alkenyl
and alkynyl groups, whether alone or as part of another group, can
be optionally substituted with A1 as defined herein.
[0315] In some embodiments, the -D moieties are the same. In yet
another embodiment, the -D moieties are different.
[0316] In one aspect, Spacer units (--Y.sub.y--) are represented by
Formulas (X)-(XII):
##STR00019##
[0317] wherein Q is --C.sub.1-C.sub.8 alkyl, --C.sub.1-C.sub.8
alkenyl, --C.sub.1-C.sub.8 alkynyl, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.1-C.sub.8 alkenyl), --O--(C.sub.1-C.sub.8 alkynyl),
-halogen, -nitro or -cyano; and m is an integer ranging from 0-4.
The akyl, alkenyl and alkynyl groups, whether alone or as part of
another group, can be optionally substituted with A1 as defined
herein.
##STR00020##
[0318] Embodiments of the Formula I and II comprising Ligand-drug
conjugate compounds can include:
##STR00021##
[0319] wherein w and y are each 0, 1 or 2, [0320] and,
##STR00022##
[0321] wherein w and y are each 0,
##STR00023##
The Drug Unit
[0322] The drug moiety (D) can be any cytotoxic, cytostatic or
immunomodulatory (e.g., immunosuppressive) or drug. D is a Drug
unit (moiety) having an atom that can form a bond with the Spacer
unit, with the Amino Acid unit, with the Stretcher unit or with the
Ligand unit. In some embodiments, the Drug unit D has a nitrogen
atom that can form a bond with the Spacer unit. As used herein, the
terms "drug unit" and "drug moiety" are synonymous and used
interchangeably.
[0323] Useful classes of cytotoxic or immunomodulatory agents
include, for example, antitubulin agents, auristatins, DNA minor
groove binders, DNA replication inhibitors, alkylating agents
(e.g., platinum complexes such as cis-platin, mono(platinum),
bis(platinum) and tri-nuclear platinum complexes and carboplatin),
anthracyclines, antibiotics, antifolates, antimetabolites,
chemotherapy sensitizers, duocarmycins, etoposides, fluorinated
pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols,
pre-forming compounds, purine antimetabolites, puromycins,
radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,
vinca alkaloids, or the like.
[0324] Individual cytotoxic or immunomodulatory agents include, for
example, an androgen, anthramycin (AMC), asparaginase,
5-azacytidine, azathioprine, bleomycin, busulfan, buthionine
sulfoximine, calicheamicin, camptothecin, carboplatin, carmustine
(BSNU), CC-1065, chlorambucil, cisplatin, colchicine,
cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B,
dacarbazine, dactinomycin (formerly actinomycin), daunorubicin,
decarbazine, docetaxel, doxorubicin, etoposide, an estrogen,
5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D,
hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU),
maytansine, mechlorethamine, melphalan, 6-mercaptopurine,
methotrexate, mithramycin, mitomycin C, mitoxantrone,
nitroimidazole, paclitaxel, palytoxin, plicamycin, procarbizine,
rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA,
topotecan, vinblastine, vincristine, vinorelbine, VP-16 and
VM-26.
[0325] In some typical embodiments, suitable cytotoxic agents
include, for example, DNA minor groove binders (e.g., enediynes and
lexitropsins, a CBI compound; see also U.S. Pat. No. 6,130,237),
duocarmycins, taxanes (e.g., paclitaxel and docetaxel), puromycins,
vinca alkaloids, CC-1065, SN-38, topotecan, morpholino-doxorubicin,
rhizoxin, cyanomorpholino-doxorubicin, echinomycin, combretastatin,
netropsin, epothilone A and B, estramustine, cryptophysins,
cemadotin, maytansinoids, discodermolide, eleutherobin, and
mitoxantrone.
[0326] In some embodiments, the Drug is an anti-tubulin agent.
Examples of anti-tubulin agents include, but are not limited to,
taxanes (e.g., Taxol.RTM. (paclitaxel), Taxotere.RTM. (docetaxel)),
T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine,
vindesine, and vinorelbine). Other antitubulin agents include, for
example, baccatin derivatives, taxane analogs (e.g., epothilone A
and B), nocodazole, colchicine and colcimid, estramustine,
cryptophycins, cemadotin, maytansinoids, combretastatins,
discodermolide, and eleutherobin.
[0327] In certain embodiments, the cytotoxic agent is a
maytansinoid, another group of anti-tubulin agents. For example, in
specific embodiments, the maytansinoid is maytansine or DM-1
(ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.
52:127-131).
[0328] In some embodiments, the Drug is an auristatin, such as
auristatin E (also known in the art as dolastatin-10) or a
derivative thereof. Typically, the auristatin E derivative is,
e.g., an ester formed between auristatin E and a keto acid. For
example, auristatin E can be reacted with paraacetyl benzoic acid
or benzoylvaleric acid to produce AEB and AEVB, respectively. Other
typical auristatin derivatives include AFP, MMAF, and MMAE. The
synthesis and structure of auristatin derivatives are described in
U.S. Patent Application Publication Nos. 2003-0083263, 2005-0238649
and 2005-0009751; International Patent Publication No. WO
04/010957, International Patent Publication No. WO 02/088172, and
U.S. Pat. Nos. 6,323,315; 6,239,104; 6,034,065; 5,780,588;
5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097;
5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988;
4,978,744; 4,879,278; 4,816,444; and 4,486,414, each of which is
incorporated by refernence herein in its entirety and for all
purposes.
[0329] Auristatins have been shown to interfere with microtubule
dynamics and nuclear and cellular division and have anticancer
activity. Auristatins of the present invention bind tubulin and can
exert a cytotoxic or cytostatic effect on a CD19 expressing cell
line. There are a number of different assays, known in the art,
that can be used for determining whether an auristatin or resultant
antibody-drug conjugate exerts a cytostatic or cytotoxic effect on
a desired cell line, see e.g., Example 7.
[0330] Methods for determining whether a compound binds tubulin are
known in the art. See, for example, Muller et al., Anal. Chem 2006,
78, 4390-4397; Hamel et al., Molecular Pharmacology, 1995 47:
965-976; and Hamel et al., The Journal of Biological Chemistry,
1990 265:28, 17141-17149. For purposes of the present invention,
the relative affinity of a compound to tubulin can be determined.
Preferred auristatins of the present invention bind tubulin with an
affinity ranging from 10 fold lower that the binding affinity of
MMAE to tubulin to 10 fold, 20 fold or even 100 fold higher than
the binding affinity of MMAE to tublin.
[0331] In some embodiments, -D is an auristatin of the formula
D.sub.E, D.sub.F or D.sub.Z:
##STR00024##
or a pharmaceutically acceptable salt or solvate form thereof;
wherein, independently at each location:
[0332] R.sup.2 is C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
or C.sub.2-C.sub.20 alkynyl;
[0333] R.sup.3 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, carbocycle, --C.sub.1-C.sub.20
alkylene (carbocycle), --C.sub.2-C.sub.20 alkenylene(carbocycle),
--C.sub.2-C.sub.20 alkynylene(carbocycle), aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle);
[0334] R.sup.4 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, carbocycle, --C.sub.1-C.sub.20
alkylene (carbocycle), --C.sub.2-C.sub.20 alkenylene(carbocycle),
--C.sub.2-C.sub.20 alkynylene(carbocycle), aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle);
[0335] R.sup.5 is H or C.sub.1-C.sub.8 alkyl;
[0336] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.s-- wherein R.sup.a and
R.sup.b are independently H, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, or carbocycle
and s is 2, 3, 4, 5 or 6,
[0337] R.sup.6 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl;
[0338] R.sup.7 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, carbocycle, --C.sub.1-C.sub.20
alkylene (carbocycle), --C.sub.2-C.sub.20 alkenylene(carbocycle),
--C.sub.2-C.sub.20 alkynylene(carbocycle), aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle);
[0339] each R.sup.8 is independently H, OH, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl,
--O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl),
--O--(C.sub.1-C.sub.20 alkynyl), or carbocycle;
[0340] R.sup.9 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl;
[0341] R.sup.24 is aryl, heterocycle, or carbocycle;
[0342] R.sup.25 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, carbocycle,
--O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl),
--O--(C.sub.2-C.sub.20 alkynyl), or OR.sup.18 wherein R.sup.18 is
H, a hydroxyl protecting group, or a direct bond where OR.sup.18
represents .dbd.O;
[0343] R.sup.26 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl, aryl, heterocycle, or
carbocycle;
[0344] R.sup.10 is aryl or heterocycle;
[0345] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl;
[0346] R.sup.11 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, carbocycle, --C.sub.1-C.sub.20
alkylene (carbocycle), --C.sub.2-C.sub.20 alkenylene(carbocycle),
--C.sub.2-C.sub.20 alkynylene(carbocycle), aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), --C.sub.2-C.sub.20 alkynylene(heterocycle)
--(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--C.sub.H(R.sup.15).sub.2;
[0347] m is an integer ranging from 1-1000;
[0348] R.sup.13 is C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20
alkenylene, or C.sub.2-C.sub.20 alkynylene;
[0349] R.sup.14 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl;
[0350] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.20 alkyl,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkenyl, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkynyl;
[0351] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl or --(CH.sub.2).sub.n--COOH;
[0352] n is an integer ranging from 0 to 6;
[0353] R.sup.27 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, O--(C.sub.1-C.sub.20 alkyl),
--O--(C.sub.2-C.sub.20 alkenyl), --O--(C.sub.2-C.sub.20 alkynyl),
halogen, --NO.sub.2, --COOH, or --C(O)OR.sup.28 wherein R.sup.28 is
H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, aryl, heterocycle,
--(CH.sub.2CH.sub.2O).sub.r--H,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.3, or
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2CH.sub.2C(O)OH; wherein r is
an integer ranging from 1-10; and
[0354] X is --(CR.sup.29.sub.2).sub.I-- wherein R.sup.29 is H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl and I is an integer ranging from 0 to 10; wherein said
alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene, aryl,
carbocyle, and heterocycle radicals, whether alone or as part of
another group, are optionally substituted.
[0355] Auristatins of the formula D.sub.E, D.sub.F or D.sub.Z
include those wherein
[0356] R.sup.2 is C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
or C.sub.2-C.sub.20 alkynyl, each of which is optionally
substituted with one or more groups independently selected from
A1;
[0357] R.sup.3 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6
carbocycle, --C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkenylene(monocyclic
C.sub.3-C.sub.6 carbocycle), --C.sub.2-C.sub.20
alkynylene(monocyclic C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10
aryl, --C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, and alkynylene radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A1, said carbocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A2,
said aryl radicals whether alone or as part of another group are
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A4;
[0358] R.sup.4 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6
carbocycle, --C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkenylene(monocyclic
C.sub.3-C.sub.6 carbocycle), --C.sub.2-C.sub.20
alkynylene(monocyclic C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10
aryl, --C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, and alkynylene radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A1, said carbocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A2,
said aryl radicals whether alone or as part of another group are
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A4;
[0359] R.sup.5 is H or C.sub.1-C.sub.8 alkyl;
[0360] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.s-- wherein R.sup.a and
R.sup.b are independently H, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, or carbocycle, and s is 2, 3, 4,
5 or 6;
[0361] R.sup.6 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl, wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted with one or more
groups independently selected from A1;
[0362] R.sup.7 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6
carbocycle, --C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkenylene(monocyclic
C.sub.3-C.sub.6 carbocycle), --C.sub.2-C.sub.20
alkynylene(monocyclic C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10
aryl, --C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, and alkynylene radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A1, said carbocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A2,
said aryl radicals whether alone or as part of another group are
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A4;
[0363] each R.sup.8 is independently H, OH, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl,
--O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl),
--O--(C.sub.1-C.sub.20 alkynyl), or carbocycle, wherein said alkyl,
alkenyl, and alkynyl radicals, whether alone or as part of another
group, are optionally substituted with one or more groups
independently selected from A1 and said carbocycle is optionally
substituted with one or more groups independently selected from
A2;
[0364] R.sup.9 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl; wherein said alkyl, alkenyl
and alkynyl radical are optionally substituted with one or more
groups independently selected from A1;
[0365] R.sup.24 is aryl, heterocycle, or carbocycle; wherein said
carbocycle radical is optionally substituted with one or more
groups independently selected from A2, said aryl radical is
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radical is optionally
substituted with one or more groups independently selected from
A4;
[0366] R.sup.25 is selected from H, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, carbocycle, OH,
--O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl),
--O--(C.sub.2-C.sub.20 alkynyl) or OR.sup.18; wherein said alkyl,
alkenyl, and alkynyl radicals, whether alone or as part of another
group, are optionally substituted with one or more groups
independently selected from A1, and said carbocycle is optionally
substituted with one or more groups independently selected from
A2;
[0367] R.sup.18 is H, a hydroxyl protecting group, or a direct bond
where OR.sup.18 represents .dbd.O;
[0368] R.sup.26 is selected from H, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, or carbocycle;
wherein said alkyl, alkenyl, and alkynyl radicals are optionally
substituted with one or more groups independently selected from A1,
and said carbocycle radical is optionally substituted with one or
more groups independently selected from A2;
[0369] R.sup.10 is aryl optionally substituted with one or more
groups independently selected from A3, or heterocycle optionally
substituted with one or more groups independently selected from
A4;
[0370] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl, each of which is optionally substituted
with one or more groups independently selected from A1;
[0371] R.sup.11 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, carbocycle, --C.sub.1-C.sub.20
alkylene (carbocycle), --C.sub.2-C.sub.20 alkenylene(carbocycle),
--C.sub.2-C.sub.20 alkynylene(carbocycle), aryl, --C.sub.1-C.sub.20
alkylene(aryl), --C.sub.2-C.sub.20 alkenylene(aryl),
--C.sub.2-C.sub.20 alkynylene(aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), --C.sub.2-C.sub.20 alkynylene(heterocycle)
--(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--C.sub.H(R.sup.15).sub.2 wherein said alkyl,
alkenyl and alkynyl radicals are optionally substituted with one or
more groups independently selected from A1, said carbocycle radical
is optionally substituted with one or more groups independently
selected from A2, said aryl radical is optionally substituted with
one or more groups independently selected from A3, and said
heterocycle is optionally substituted with one or more groups
independently selected from A4;
[0372] m is an integer ranging from 1-1000;
[0373] R.sup.13 is C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20
alkenylene, or C.sub.2-C.sub.20 alkynylene, each of which is
optionally substituted with one or more groups independently
selected from A1;
[0374] R.sup.14 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted with one or more
groups independently selected from A1;
[0375] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.20 alkyl,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkenyl, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkynyl wherein said
alkyl, alkenyl and alkynyl radicals are optionally substituted with
one or more groups independently selected from A1;
[0376] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl or --(CH.sub.2).sub.n--COOH wherein said alkyl, alkenyl and
alkynyl radicals are optionally substituted with one or more groups
independently selected from A1;
[0377] n is an integer ranging from 0 to 6;
[0378] R.sup.27 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, O--(C.sub.1-C.sub.20 alkyl),
--O--(C.sub.2-C.sub.20 alkenyl), --O--(C.sub.2-C.sub.20 alkynyl),
halogen, --NO.sub.2, --COOH, or --C(O)OR.sup.28 wherein said alkyl,
alkenyl and alkynyl radicals are optionally substituted with one or
more groups independently selected from A1 and R.sup.28 is H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl, aryl, heterocycle, --(CH.sub.2CH.sub.2O).sub.r--H,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.3, or
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.2CH.sub.2C(O)OH; wherein r is
an integer ranging from 1-10 and wherein said alkyl, alkenyl and
alkynyl radicals are optionally substituted with one or more groups
independently selected from A1; said aryl radical is optionally
substituted with one or more groups independently selected from A3;
and said heterocycle radical is optionally substituted with one or
more groups independently selected from A4; and
[0379] X is --(CR.sup.29.sub.2).sub.I-- wherein R.sup.29 is H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl and I is an integer ranging from 0 to 10 and wherein said
alkyl, alkenyl and alkynyl radicals are optionally substituted with
one or more groups independently selected from A1;
[0380] A1 is halogen, optionally substituted --O--(C.sub.1-C.sub.8
alkyl), optionally substituted --O--(C.sub.2-C.sub.8 alkenyl),
optionally substituted --O--(C.sub.2-C.sub.8 alkynyl), optionally
substituted -aryl, --C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2,
--C(O)NHR', --C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R',
--S(O).sub.2R', --S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2,
--NH(R'), --N(R').sub.2 and --CN, where each R' is independently
selected from H, optionally substituted --C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, or optionally substituted
aryl, and wherein said optionally substituted O--(C.sub.1-C.sub.8
alkyl), optionally substituted --O--(C.sub.2-C.sub.8 alkenyl),
optionally substituted --O--(C.sub.2-C.sub.8 alkynyl), optionally
substituted aryl, optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, and optionally
substituted --C.sub.2-C.sub.8 alkynyl groups can be optionally
substituted with one or more groups independently selected from
--C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl;
[0381] A2 is halogen, optionally substituted C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, .dbd.O, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl and
wherein said optionally substituted --C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), and optionally substituted aryl
groups can be optionally substituted with one or more substituents
independently selected from C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, halogen,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'', --OC(O)R'',
--C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'',
--C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl;
[0382] A3 is halogen, optionally substituted --C.sub.1-C.sub.8
alkyl, optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, optionally substituted
--O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted -aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, --NO.sub.2, --N.sub.3, --NH.sub.2, --NH(R'),
--N(R').sub.2 and --CN, where each R' is independently selected
from H, optionally substituted --C.sub.1-C.sub.8 alkyl, optionally
substituted --C.sub.2-C.sub.8 alkenyl, optionally substituted
--C.sub.2-C.sub.8 alkynyl, or optionally substituted aryl and
wherein said optionally substituted --C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, optionally substituted
O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), and optionally substituted aryl,
groups can be further optionally substituted with one or more
substituents independently selected from C.sub.1-C.sub.8 alkyl,
--C.sub.2-C.sub.8 alkenyl, --C.sub.2-C.sub.8 alkynyl, halogen,
--O--(C.sub.1-C.sub.8 alkyl), --O--(C.sub.2-C.sub.8 alkenyl),
--O--(C.sub.2-C.sub.8 alkynyl), -aryl, --C(O)R'', --OC(O)R'',
--C(O)OR'', --C(O)NH.sub.2, --C(O)NHR'',
--C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl;
[0383] and A4 is optionally substituted --C.sub.1-C.sub.8 alkyl,
optionally substituted --C.sub.2-C.sub.8 alkenyl, optionally
substituted --C.sub.2-C.sub.8 alkynyl, halogen, optionally
substituted --O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted -aryl,
--C(O)R', --OC(O)R', --C(O)OR', --C(O)NH.sub.2, --C(O)NHR',
--C(O)N(R').sub.2, --NHC(O)R', --SR', --SO.sub.3R', --S(O).sub.2R',
--S(O)R', --OH, --N.sub.3, --NH.sub.2, --NH(R'), --N(R').sub.2 and
--CN, where each R' is independently selected from H, optionally
substituted --C.sub.1-C.sub.8 alkyl, optionally substituted
--C.sub.2-C.sub.8 alkenyl, optionally substituted --C.sub.2-C.sub.8
alkynyl, or optionally substituted aryl and wherein said optionally
substituted O--(C.sub.1-C.sub.8 alkyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkenyl), optionally substituted
--O--(C.sub.2-C.sub.8 alkynyl), optionally substituted
--C.sub.1-C.sub.8 alkyl, optionally substituted --C.sub.2-C.sub.8
alkenyl, optionally substituted --C.sub.2-C.sub.8 alkynyl, and
optionally substituted aryl groups can be further optionally
substituted with one or more substituents independently selected
from --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, halogen, --O--(C.sub.1-C.sub.8 alkyl),
--O--(C.sub.2-C.sub.8 alkenyl), --O--(C.sub.2-C.sub.8 alkynyl),
-aryl, --C(O)R'', --OC(O)R'', --C(O)OR'', --C(O)NH.sub.2,
--C(O)NHR'', --C(O)N(R'').sub.2--NHC(O)R'', --SR'', --SO.sub.3R'',
--S(O).sub.2R'', --S(O)R'', --OH, --N.sub.3, --NH.sub.2, --NH(R''),
--N(R'').sub.2 and --CN, where each R'' is independently selected
from H, --C.sub.1-C.sub.8 alkyl, --C.sub.2-C.sub.8 alkenyl,
--C.sub.2-C.sub.8 alkynyl, or aryl; or a pharmaceutically
acceptable salt or solvate form thereof.
[0384] Auristatins of the formula D.sub.E include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted.
[0385] Auristatins of the formula D.sub.E include those wherein the
groups of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, and R.sup.9 are unsubstituted and the groups of R.sup.24,
R.sup.25 and R.sup.26 are optionally substituted as described
herein.
[0386] Auristatins of the formula D.sub.E include those wherein
[0387] R.sup.2 is C.sub.1-C.sub.20 alkyl optionally substituted
with one or more groups independently selected from A1;
[0388] R.sup.3 and R.sup.7 are independently selected from H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl, monocyclic C.sub.3-C.sub.6 carbocycle, --C.sub.1-C.sub.20
alkylene(monocyclic C.sub.3-C.sub.6 carbocycle), --C.sub.2-C.sub.20
alkenylene(monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.20 alkynylene(monocyclic C.sub.3-C.sub.6
carbocycle), C.sub.6-C.sub.10 aryl, --C.sub.1-C.sub.20
alkylene(C.sub.6-C.sub.10 aryl), --C.sub.2-C.sub.20
alkenylene(C.sub.6-C.sub.10 aryl), --C.sub.2-C.sub.20
alkynylene(C.sub.6-C.sub.10 aryl), heterocycle, --C.sub.1-C.sub.20
alkylene(heterocycle), --C.sub.2-C.sub.20 alkenylene(heterocycle),
or --C.sub.2-C.sub.20 alkynylene(heterocycle); wherein said alkyl,
alkenyl, alkynyl, alkylene, alkenylene, and alkynylene radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A1,
said carbocycle radicals whether alone or as part of another group
are optionally substituted with one or more groups independently
selected from A2, said aryl radicals whether alone or as part of
another group are optionally substituted with one or more groups
independently selected from A3, and said heterocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from
A4;
[0389] R.sup.4 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6
carbocycle, --C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkenylene(monocyclic
C.sub.3-C.sub.6 carbocycle), --C.sub.2-C.sub.20
alkynylene(monocyclic C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10
aryl, --C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, and alkynylene radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A1, said carbocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A2,
said aryl radicals whether alone or as part of another group are
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A4;
[0390] R.sup.5 is H or C.sub.1-C.sub.8 alkyl;
[0391] or R.sup.4 and R.sup.5 jointly form a carbocyclic ring and
have the formula --(CR.sup.aR.sup.b).sub.s-- wherein R.sup.a and
R.sup.b are independently selected from H, C.sub.1-C.sub.8 alkyl,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, or carbocycle,
and s is selected from 2, 3, 4, 5 or 6;
[0392] R.sup.6 is C.sub.1-C.sub.20 alkyl optionally substituted
with one or more groups independently selected from A1;
[0393] each R.sup.8 is independently selected from OH,
O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl), or
--O--(C.sub.2-C.sub.20 alkynyl) wherein said alkyl, alkenyl, and
alkynyl radicals are optionally substituted with one or more groups
independently selected from A1;
[0394] R.sup.9 is hydrogen or C.sub.1-C.sub.20 alkyl optionally
substituted with one or more groups independently selected from
A1;
[0395] R.sup.24 is aryl, heterocycle, or carbocycle; wherein said
carbocycle radical is optionally substituted with one or more
groups independently selected from A2, said aryl radical is
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radical is optionally
substituted with one or more groups independently selected from
A4;
[0396] R.sup.25 is OR.sup.18; wherein R.sup.18 is H, a hydroxyl
protecting group, or a direct bond where OR.sup.18 represents
.dbd.O;
[0397] R.sup.26 is selected from H, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, or carbocycle;
wherein said alkyl, alkenyl, and alkynyl radicals are optionally
substituted with one or more groups independently selected from A1,
and said carbocycle radical is optionally substituted with one or
more groups independently selected from A2;
and A1, A2, A3, and A4 are as defined herein; or a pharmaceutically
acceptable salt or solvate form thereof.
[0398] Auristatins of the formula D.sub.E include those wherein
[0399] R.sup.2 is C.sub.1-C.sub.8 alkyl;
[0400] R.sup.3, R.sup.4 and R.sup.7 are independently selected from
H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6 carbocycle,
--C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.20 alkenylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkynylene(monocyclic
C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10 aryl,
--C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, and alkynylene radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A1, said carbocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A2,
said aryl radicals whether alone or as part of another group are
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A4;
[0401] R.sup.5 is hydrogen;
[0402] R.sup.6 is C.sub.1-C.sub.8 alkyl;
[0403] each R.sup.8 is independently selected from OH,
O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20 alkenyl), or
--O--(C.sub.2-C.sub.20 alkynyl) wherein said alkyl, alkenyl, and
alkynyl radicals are optionally substituted with one or more groups
independently selected from A1;
[0404] R.sup.9 is hydrogen or C.sub.1-C.sub.8 alkyl;
[0405] R.sup.24 is phenyl optionally substituted with one or more
groups independently selected from A3;
[0406] R.sup.25 is OR.sup.18; wherein R.sup.18 is H, a hydroxyl
protecting group, or a direct bond where OR.sup.18 represents
.dbd.O;
[0407] R.sup.26 is selected from H, C.sub.1-C.sub.20 alkyl,
C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20 alkynyl, or carbocycle;
wherein said alkyl, alkenyl, and alkynyl radicals are optionally
substituted with one or more groups independently selected from A1,
and said carbocycle radical is optionally substituted with one or
more groups independently selected from A2; and
A1, A2, A3, and A4 are as defined herein; or a pharmaceutically
acceptable salt or solvate form thereof.
[0408] Auristatins of the formula D.sub.E include those wherein
[0409] R.sup.2 is methyl;
[0410] R.sup.3 is H, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8
alkenyl, or C.sub.2-C.sub.8 alkynyl, wherein said alkyl, alkenyl
and alkynyl radicals are optionally optionally substituted with one
or more groups independently selected from A1;
[0411] R.sup.4 is H, C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.8 alkynyl, monocyclic C.sub.3-C.sub.6
carbocycle, C.sub.6-C.sub.10 aryl, --C.sub.1-C.sub.8
alkylene(C.sub.6-C.sub.10 aryl), C.sub.2-C.sub.8
alkenylene(C.sub.6-C.sub.10 aryl), --C.sub.2-C.sub.8
alkynylene(C.sub.6-C.sub.10 aryl), --C.sub.1-C.sub.8 alkylene
(monocyclic C.sub.3-C.sub.6 carbocycle), --C.sub.2-C.sub.8
alkenylene (monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.8 alkynylene(monocyclic C.sub.3-C.sub.6
carbocycle); wherein said alkyl, alkenyl, alkynyl, alkylene,
alkenylene, and alkynylene radicals whether alone or as part of
another group are optionally substituted with one or more groups
independently selected from A1; said carbocyle radicals whether
alone or as part of another group are optionally substituted with
one or more groups independently selected from A2; and said aryl
radicals whether alone or as part of another group are optionally
substituted with one or more groups independently selected from
A3;
[0412] R.sup.5 is H; R.sup.6 is methyl;
[0413] R.sup.7 is C.sub.1-C.sub.8 alkyl, C.sub.2-C.sub.8 alkenyl or
C.sub.2-C.sub.8 alkynyl;
[0414] each R.sup.8 is methoxy;
[0415] R.sup.9 is hydrogen or C.sub.1-C.sub.8 alkyl;
[0416] R.sup.24 is phenyl;
[0417] R.sup.25 is OR.sup.18; wherein R.sup.18 is H, a hydroxyl
protecting group, or a direct bond where OR.sup.18 represents
.dbd.O;
[0418] R.sup.26 is methyl; and A1, A2, and A3 are as defined
herein; or a pharmaceutically acceptable salt or solvate form
thereof.
[0419] Auristatins of the formula D.sub.E include those wherein
R.sup.2 is methyl; R.sup.3 is H or C.sub.1-C.sub.3 alkyl; R.sup.4
is C.sub.1-C.sub.5 alkyl; R.sup.5 is H; R.sup.6 is methyl; R.sup.7
is isopropyl or sec-butyl; R.sup.8 is methoxy; R.sup.9 is hydrogen
or C.sub.1-C.sub.8 alkyl; R.sup.24 is phenyl; R.sup.25 is
OR.sup.18; wherein R.sup.18 is H, a hydroxyl protecting group, or a
direct bond where OR.sup.18 represents .dbd.O; and R.sup.26 is
methyl; or a pharmaceutically acceptable salt or solvate form
thereof.
[0420] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein
[0421] R.sup.2 is C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
or C.sub.2-C.sub.20 alkynyl, each of which is optionally
substituted with one or more groups independently selected from
A1;
[0422] R.sup.3, R.sup.4, and R.sup.7 are independently selected
from H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6 carbocycle,
--C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.20 alkenylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkynylene(monocyclic
C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10 aryl,
--C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, and alkynylene radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A1, said carbocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A2,
said aryl radicals whether alone or as part of another group are
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A4;
[0423] R.sup.5 is H;
[0424] R.sup.6 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl, wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted with one or more
groups independently selected from A1;
[0425] each R.sup.8 is independently selected from H, OH,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl, --O--(C.sub.1-C.sub.20 alkyl), --O--(C.sub.2-C.sub.20
alkenyl), --O--(C.sub.1-C.sub.20 alkynyl), or carbocycle, wherein
said alkyl, alkenyl, and alkynyl radicals, whether alone or as part
of another group, are optionally substituted with one or more
groups independently selected from A1 and said carbocycle is
optionally substituted with one or more groups independently
selected from A2;
[0426] R.sup.9 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl; wherein said alkyl, alkenyl
and alkynyl radical are optionally substituted with one or more
groups independently selected from A1;
[0427] R.sup.10 is phenyl optionally substituted with one or more
groups independently selected from A3;
[0428] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl, each of which is optionally substituted
with one or more groups independently selected from A1;
[0429] R.sup.11 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, aryl, heterocycle,
--(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2 wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted with one or more
groups independently selected from A1, said aryl radical is
optionally substituted with one or more groups independently
selected from A3, and said heterocycle is optionally substituted
with one or more groups independently selected from A4;
[0430] m is an integer ranging from 1-1000;
[0431] R.sup.13 is C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20
alkenylene, or C.sub.2-C.sub.20 alkynylene, each of which is
optionally substituted with one or more groups independently
selected from A1;
[0432] R.sup.14 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted with one or more
groups independently selected from A1;
[0433] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.20 alkyl,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkenyl, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkynyl wherein said
alkyl, alkenyl and alkynyl radicals are optionally substituted with
one or more groups independently selected from A1;
[0434] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl or --(CH.sub.2).sub.n--COOH wherein said alkyl, alkenyl and
alkynyl radicals are optionally substituted with one or more groups
independently selected from A1;
[0435] n is an integer ranging from 0 to 6;
[0436] R.sup.27 is H; and
[0437] X is --(CR.sup.29.sub.2).sub.I-- wherein I is 0; and A1, A2,
A3, and A4 are as defined herein; or a pharmaceutically acceptable
salt or solvate form thereof.
[0438] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein
[0439] R.sup.2 is methyl;
[0440] R.sup.3, R.sup.4, and R.sup.7 are independently selected
from H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl,
C.sub.2-C.sub.20 alkynyl, monocyclic C.sub.3-C.sub.6 carbocycle,
--C.sub.1-C.sub.20 alkylene(monocyclic C.sub.3-C.sub.6 carbocycle),
--C.sub.2-C.sub.20 alkenylene(monocyclic C.sub.3-C.sub.6
carbocycle), --C.sub.2-C.sub.20 alkynylene(monocyclic
C.sub.3-C.sub.6 carbocycle), C.sub.6-C.sub.10 aryl,
--C.sub.1-C.sub.20 alkylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkenylene(C.sub.6-C.sub.10 aryl),
--C.sub.2-C.sub.20 alkynylene(C.sub.6-C.sub.10 aryl), heterocycle,
--C.sub.1-C.sub.20 alkylene(heterocycle), --C.sub.2-C.sub.20
alkenylene(heterocycle), or --C.sub.2-C.sub.20
alkynylene(heterocycle); wherein said alkyl, alkenyl, alkynyl,
alkylene, alkenylene, and alkynylene radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A1, said carbocycle radicals
whether alone or as part of another group are optionally
substituted with one or more groups independently selected from A2,
said aryl radicals whether alone or as part of another group are
optionally substituted with one or more groups independently
selected from A3, and said heterocycle radicals whether alone or as
part of another group are optionally substituted with one or more
groups independently selected from A4;
[0441] R.sup.5 is H;
[0442] R.sup.6 is methyl;
[0443] each R.sup.8 is methoxy;
[0444] R.sup.9 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl; wherein said alkyl, alkenyl
and alkynyl radical are optionally substituted with one or more
groups independently selected from A1;
[0445] R.sup.10 is aryl optionally substituted with one or more
groups independently selected from A3, or heterocycle optionally
substituted with one or more groups independently selected from
A4;
[0446] Z is O, S, NH, or NR.sup.12, wherein R.sup.12 is
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, or
C.sub.2-C.sub.20 alkynyl, each of which is optionally substituted
with one or more groups independently selected from A1;
[0447] R.sup.11 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, C.sub.2-C.sub.20 alkynyl, aryl, heterocycle,
--(R.sup.13O).sub.m--R.sup.14, or
--(R.sup.13O).sub.m--CH(R.sup.15).sub.2 wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted with one or more
groups independently selected from A1, said aryl radical is
optionally substituted with one or more groups independently
selected from A3, and said heterocycle is optionally substituted
with one or more groups independently selected from A4;
[0448] m is an integer ranging from 1-1000;
[0449] R.sup.13 is C.sub.2-C.sub.20 alkylene, C.sub.2-C.sub.20
alkenylene, or C.sub.2-C.sub.20 alkynylene, each of which is
optionally substituted with one or more groups independently
selected from A1;
[0450] R.sup.14 is H, C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20
alkenyl, or C.sub.2-C.sub.20 alkynyl wherein said alkyl, alkenyl
and alkynyl radicals are optionally substituted with one or more
groups independently selected from A1;
[0451] each occurrence of R.sup.15 is independently H, COOH,
--(CH.sub.2).sub.n--N(R.sup.16).sub.2,
--(CH.sub.2).sub.n--SO.sub.3H,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.1-C.sub.20 alkyl,
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkenyl, or
--(CH.sub.2).sub.n--SO.sub.3--C.sub.2-C.sub.20 alkynyl wherein said
alkyl, alkenyl and alkynyl radicals are optionally substituted with
one or more groups independently selected from A1;
[0452] each occurrence of R.sup.16 is independently H,
C.sub.1-C.sub.20 alkyl, C.sub.2-C.sub.20 alkenyl, C.sub.2-C.sub.20
alkynyl or --(CH.sub.2).sub.n--COOH wherein said alkyl, alkenyl and
alkynyl radicals are optionally substituted with one or more groups
independently selected from A1;
[0453] n is an integer ranging from 0 to 6;
[0454] R.sup.27 is H; and
[0455] X is --(CR.sup.29.sub.2).sub.I-- wherein I is 0; and A1, A2,
A3, and A4 are as defined herein; or a pharmaceutically acceptable
salt or solvate form thereof.
[0456] In certain of these embodiments, R.sup.10 is phenyl
optionally substituted with one or more groups independently
selected from A3.
[0457] Auristatins of the formula D.sub.F include those wherein the
groups of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, and R.sup.9 are unsubstituted and the groups of R.sup.10
and R.sup.11 are as described herein.
[0458] Auristatins of the formula D.sub.F include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted
[0459] Auristatins of the formula D.sub.F include those wherein
[0460] R.sup.2 is methyl; R.sup.3 is H or C.sub.1-C.sub.3 alkyl;
R.sup.4 is C.sub.1-C.sub.5 alkyl; R.sup.5 is H; R.sup.6 is methyl;
R.sup.7 is isopropyl or sec-butyl; R.sup.8 is methoxy; R.sup.9 is
hydrogen or C.sub.1-C.sub.8 alkyl; R.sup.10 is phenyl optionally
substituted with one or more groups independently selected from A3;
Z is O, S, or NH; R.sup.11 is as defined herein; or a
pharmaceutically acceptable salt or solvate form thereof.
[0461] Auristatins of the formula D.sub.F include those wherein
[0462] R.sup.1 is hydrogen; R.sup.2 is methyl; R.sup.3 is H or
C.sub.1-C.sub.3 alkyl; R.sup.4 is C.sub.1-C.sub.5 alkyl; R.sup.5 is
H; R.sup.6 is methyl; R.sup.7 is isopropyl or sec-butyl; R.sup.8 is
methoxy; R.sup.9 is hydrogen or C.sub.1-C.sub.8 alkyl; R.sup.10 is
phenyl; Z is O or NH; R.sup.11 is as defined herein; or a
pharmaceutically acceptable salt or solvate form thereof.
[0463] Auristatins of the formula D.sub.Z include those wherein the
groups of R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7,
R.sup.8, R.sup.9, R.sup.27, R.sup.28, and R.sup.29 are
unsubstituted and the groups of R.sup.10 and R.sup.11 are as
described herein.
[0464] Auristatins of the formula D.sub.Z include those wherein
said alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynyklene,
aryl, carbocyle, and heterocycle radicals are unsubstituted
[0465] Auristatins of the formula D.sub.Z include those wherein
[0466] R.sup.1 is hydrogen; R.sup.2 is methyl; R.sup.3 is H or
C.sub.1-C.sub.3 alkyl; R.sup.4 is C.sub.1-C.sub.5 alkyl; R.sup.5 is
H; R.sup.6 is methyl; R.sup.7 is isopropyl or sec-butyl; R.sup.8 is
methoxy; R.sup.9 is hydrogen or C.sub.1-C.sub.8 alkyl;
R.sup.10 is phenyl optionally substituted with one or more groups
independently selected from A3; Z is O, S, or NH; R.sup.11 is as
defined herein; R.sup.27 is H; and X is --(CR.sup.29.sub.2).sub.I--
wherein I is 0; or a pharmaceutically acceptable salt or solvate
form thereof.
[0467] Auristatins of the formula D.sub.Z include those wherein
[0468] R.sup.1 is hydrogen; R.sup.2 is methyl; R.sup.3 is H or
C.sub.1-C.sub.3 alkyl; R.sup.4 is C.sub.1-C.sub.5 alkyl; R.sup.5 is
H; R.sup.6 is methyl; R.sup.7 is isopropyl or sec-butyl; R.sup.8 is
methoxy; R.sup.9 is hydrogen or C.sub.1-C.sub.8 alkyl; R.sup.10 is
phenyl; R.sup.11 is as defined herein; R.sup.27 is H; X is
--(CR.sup.29.sub.2).sub.I-- wherein I is 0; and Z is O or NH; or a
pharmaceutically acceptable salt or solvate form thereof.
[0469] Auristatins of the formula D.sub.Z include those wherein
R.sup.11 is --(CH.sub.2CH.sub.2O).sub.r--H,
--(CH.sub.2CH.sub.2O).sub.r--CH.sub.3, or
--(CH.sub.2CH.sub.2).sub.r--CH.sub.2CH.sub.2C(O)OH; wherein r is an
integer ranging from 1-10; or a pharmaceutically acceptable salt or
solvate form thereof.
[0470] Auristatins of the formula D.sub.Z include those wherein the
phenyl group at the amino terminus is para substituted as shown
below:
##STR00025##
[0471] Auristatins of the formula D.sub.E, D.sub.F or D.sub.Z
include those wherein R.sup.3, R.sup.4 and R.sup.7 are
independently isopropyl or sec-butyl and R.sup.5 is --H. In an
exemplary embodiment, R.sup.3 and R.sup.4 are each isopropyl,
R.sup.5 is H, and R.sup.7 is sec-butyl. The remainder of the
substituents are as defined herein.
[0472] Auristatins of the formula D.sub.E, D.sub.F or D.sub.Z
include those wherein R.sup.2 and R.sup.6 are each methyl, and
R.sup.9 is H. The remainder of the substituents are as defined
herein.
[0473] Auristatins of the formula D.sub.E, D.sub.F or D.sub.Z
include those wherein each occurrence of R.sup.8 is --OCH.sub.3.
The remainder of the substituents are as defined herein.
[0474] Auristatins of the formula D.sub.E, D.sub.F or D.sub.Z
include those wherein R.sup.3 and R.sup.4 are each isopropyl,
R.sup.2 and R.sup.6 are each methyl, R.sup.5 is H, R.sup.7 is
sec-butyl, each occurrence of R.sup.8 is --OCH.sub.3, and R.sup.9
is H. The remainder of the substituents are as defined herein.
[0475] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein Z is --O-- or --NH--. The remainder of the substituents are
as defined herein.
[0476] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein R.sup.10 is aryl. The remainder of the substituents are as
defined herein.
[0477] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein, R.sup.10 is -phenyl. The remainder of the substituents are
as defined herein.
[0478] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein Z is --O--, and R.sup.11 is H, methyl or t-butyl. The
remainder of the substituents are as defined herein.
[0479] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein, when Z is --NH, R.sup.11 is --CH(R.sup.15).sub.2, wherein
R.sup.15 is --(CH.sub.2).sub.n--N(R.sup.16).sub.2, and R.sup.16 is
--C.sub.1-C.sub.8 alkyl or --(CH.sub.2).sub.n--COOH. The remainder
of the substituents are as defined herein.
[0480] Auristatins of the formula D.sub.F or D.sub.Z include those
wherein when Z is --NH, R.sup.11 is --CH(R.sup.15).sub.2, wherein
R.sup.15 is --(CH.sub.2).sub.n--SO.sub.3H. The remainder of the
substituents are as defined herein.
[0481] Illustrative Drug units (-D) include the drug units having
the following structures:
##STR00026## ##STR00027## ##STR00028##
or pharmaceutically acceptable salts or solvates thereof.
[0482] In one aspect, hydrophilic groups, such as but not limited
to triethylene glycol esters (TEG) can be attached to the Drug Unit
at R.sup.11. Without being bound by theory, the hydrophilic groups
assist in the internalization and non-agglomeration of the Drug
Unit.
[0483] In some embodiments, the Drug unit is not TZT-1027. In some
embodiments, the Drug unit is not auristatin E, dolastatin 10, or
auristatin PE.
[0484] Exemplary ligand-drug conjugate compounds have the following
structures wherein "mAb-s-" represents an anti-CD19 antibody:
##STR00029## ##STR00030##
[0485] or pharmaceutically acceptable salt or solvate forms
thereof, wherein Val is valine, and Cit is citrulline.
[0486] In certain embodiments, the Drug is an antimetabolite. The
antimetabolite can be, for example, a purine antagonist (e.g.,
azothioprine or mycophenolate mofetil), a dihydrofolate reductase
inhibitor (e.g., methotrexate), acyclovir, gangcyclovir,
zidovudine, vidarabine, ribavarin, azidothymidine, cytidine
arabinoside, amantadine, dideoxyuridine, iododeoxyuridine,
poscarnet, or trifluridine.
[0487] In other embodiments, the Drug is tacrolimus, cyclosporine
or rapamycin. In further embodiments, the Drug is aldesleukin,
alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine,
anastrozole, arsenic trioxide, bexarotene, bexarotene, calusterone,
capecitabine, celecoxib, cladribine, Darbepoetin alfa, Denileukin
diftitox, dexrazoxane, dromostanolone propionate, epirubicin,
Epoetin alfa, estramustine, exemestane, Filgrastim, floxuridine,
fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin,
goserelin, idarubicin, ifosfamide, imatinib mesylate, Interferon
alfa-2a, irinotecan, letrozole, leucovorin, levamisole,
meclorethamine or nitrogen mustard, megestrol, mesna, methotrexate,
methoxsalen, mitomycin C, mitotane, nandrolone phenpropionate,
oprelvekin, oxaliplatin, pamidronate, pegademase, pegaspargase,
pegfilgrastim, pentostatin, pipobroman, plicamycin, porfimer
sodium, procarbazine, quinacrine, rasburicase, Rituximab,
Sargramostim, streptozocin, tamoxifen, temozolomide, teniposide,
testolactone, thioguanine, toremifene, Tositumomab, Trastuzumab,
tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,
vinorelbine and zoledronate.
[0488] In some embodiments, the Drug moiety is an immunomodulatory
agent. The immunomodulatory agent can be, for example, gancyclovir,
etanercept, tacrolimus, cyclosporine, rapamycin, cyclophosphamide,
azathioprine, mycophenolate mofetil or methotrexate. Alternatively,
the immunomodulatory agent can be, for example, a glucocorticoid
(e.g., cortisol or aldosterone) or a glucocorticoid analogue (e.g.,
prednisone or dexamethasone).
[0489] In some embodiments, the immunomodulatory agent is an
anti-inflammatory agent, such as arylcarboxylic derivatives,
pyrazole-containing derivatives, oxicam derivatives and nicotinic
acid derivatives. Classes of anti-inflammatory agents include, for
example, cyclooxygenase inhibitors, 5-lipoxygenase inhibitors, and
leukotriene receptor antagonists.
[0490] Suitable cyclooxygenase inhibitors include meclofenamic
acid, mefenamic acid, carprofen, diclofenac, diflunisal, fenbufen,
fenoprofen, ibuprofen, indomethacin, ketoprofen, nabumetone,
naproxen, sulindac, tenoxicam, tolmetin, and acetylsalicylic
acid.
[0491] Suitable lipoxygenase inhibitors include redox inhibitors
(e.g., catechol butane derivatives, nordihydroguaiaretic acid
(NDGA), masoprocol, phenidone, Iampalen, indazolinones,
naphazatrom, benzofuranol, alkylhydroxylamine), and non-redox
inhibitors (e.g., hydroxythiazoles, methoxyalkylthiazoles,
benzopyrans and derivatives thereof, methoxytetrahydropyran,
boswellic acids and acetylated derivatives of boswellic acids, and
quinolinemethoxyphenylacetic acids substituted with cycloalkyl
radicals), and precursors of redox inhibitors.
[0492] Other suitable lipoxygenase inhibitors include antioxidants
(e.g., phenols, propyl gallate, flavonoids and/or naturally
occurring substrates containing flavonoids, hydroxylated
derivatives of the flavones, flavonol, dihydroquercetin, luteolin,
galangin, orobol, derivatives of chalcone,
4,2',4'-trihydroxychalcone, ortho-aminophenols, N-hydroxyureas,
benzofuranols, ebselen and species that increase the activity of
the reducing selenoenzymes), iron chelating agents (e.g.,
hydroxamic acids and derivatives thereof, N-hydroxyureas,
2-benzyl-1-naphthol, catechols, hydroxylamines, carnosol trolox C,
catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic
acid and 4-(omega-arylalkyl)phenylalkanoic acids),
imidazole-containing compounds (e.g., ketoconazole and
itraconazole), phenothiazines, and benzopyran derivatives.
[0493] Yet other suitable lipoxygenase inhibitors include
inhibitors of eicosanoids (e.g., octadecatetraenoic,
eicosatetraenoic, docosapentaenoic, eicosahexaenoic and
docosahexaenoic acids and esters thereof, PGE1 (prostaglandin E1),
PGA2 (prostaglandin A2), viprostol, 15-monohydroxyeicosatetraenoic,
15-monohydroxy-eicosatrienoic and 15-monohydroxyeicosapentaenoic
acids, and leukotrienes B5, C5 and D5), compounds interfering with
calcium flows, phenothiazines, diphenylbutylamines, verapamil,
fuscoside, curcumin, chlorogenic acid, caffeic acid,
5,8,11,14-eicosatetrayenoic acid (ETYA), hydroxyphenylretinamide,
Ionapalen, esculin, diethylcarbamazine, phenantroline, baicalein,
proxicromil, thioethers, diallyl sulfide and di-(1-propenyl)
sulfide.
[0494] Leukotriene receptor antagonists include calcitriol,
ontazolast, Bayer Bay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo
Denmark ETH-615, Lilly LY-293111, Ono ONO-4057, Terumo TMK-688,
Boehringer Ingleheim BI-RM-270, Lilly LY 213024, Lilly LY 264086,
Lilly LY 292728, Ono ONO LB457, Pfizer 105696, Perdue Frederick PF
10042, Rhone-Poulenc Rorer RP 66153, SmithKline Beecham SB-201146,
SmithKline Beecham SB-201993, SmithKline Beecham SB-209247, Searle
SC-53228, Sumitamo SM 15178, American Home Products WAY 121006,
Bayer Bay-o-8276, Warner-Lambert CI-987, Warner-Lambert
CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283, MacroNex
MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, Ono
ONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG 14893,
Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698,
Shionoogi S-2474, Searle SC-41930, Searle SC-50505, Searle
SC-51146, Searle SC-52798, SmithKline Beecham SK&F-104493, Leo
Denmark SR-2566, Tanabe T-757 and Teijin TEI-1338.
[0495] In certain embodiments, the cytotoxic or cytostatic agent is
a dolastatin. In certain embodiments, the cytotoxic or cytostatic
agent is of the auristatin class. Thus, in a specific embodiment,
the cytotoxic or cytostatic agent is MMAE (Formula XI). In another
specific embodiment, the cytotoxic or cytostatic agent is AFP
(Formula XVI).
##STR00031##
[0496] In certain embodiments, the cytotoxic or cytostatic agent is
a compound of formulas XII-XXI or pharmaceutically acceptable salt
or solvate form thereof:
##STR00032## ##STR00033##
[0497] Methods of determining whether a Drug or Ligand-Drug
conjugate exerts a cytostatic and/or cytotoxic effect on a cell are
known. Generally, the cytotoxic or cytostatic activity of a Ligand
Drug conjugate can be measured by: exposing mammalian cells
expressing a target protein of the Ligand Drug conjugate in a cell
culture medium; culturing the cells for a period from about 6 hours
to about 5 days; and measuring cell viability. Cell-based in vitro
assays can be used to measure viability (proliferation),
cytotoxicity, and induction of apoptosis (caspase activation) of
the Ligand Drug conjugate.
[0498] For determining whether a Ligand Drug conjugate exerts a
cytostatic effect, a thymidine incorporation assay may be used. For
example, cancer cells expressing a target antigen at a density of
5,000 cells/well of a 96-well plated can be cultured for a 72-hour
period and exposed to 0.5 .mu.Ci of .sup.3H-thymidine during the
final 8 hours of the 72-hour period. The incorporation of
.sup.3H-thymidine into cells of the culture is measured in the
presence and absence of the Ligand Drug conjugate.
[0499] For determining cytotoxicity, necrosis or apoptosis
(programmed cell death) can be measured. Necrosis is typically
accompanied by increased permeability of the plasma membrane;
swelling of the cell, and rupture of the plasma membrane. Apoptosis
is typically characterized by membrane blebbing, condensation of
cytoplasm, and the activation of endogenous endonucleases.
Determination of any of these effects on cancer cells indicates
that a Ligand Drug conjugate is useful in the treatment of
cancers.
[0500] Cell viability can be measured by determining in a cell the
uptake of a dye such as neutral red, trypan blue, or ALAMAR.TM.
blue (see, e.g., Page et al., 1993, Intl. J. Oncology 3:473-476).
In such an assay, the cells are incubated in media containing the
dye, the cells are washed, and the remaining dye, reflecting
cellular uptake of the dye, is measured spectrophotometrically. The
protein-binding dye sulforhodamine B (SRB) can also be used to
measure cytoxicity (Skehan et al., 1990, J. Natl. Cancer Inst.
82:1107-12).
[0501] Alternatively, a tetrazolium salt, such as MTT, is used in a
quantitative colorimetric assay for mammalian cell survival and
proliferation by detecting living, but not dead, cells (see, e.g.,
Mosmann, 1983, J. Immunol. Methods 65:55-63).
[0502] Apoptosis can be quantitated by measuring, for example, DNA
fragmentation. Commercial photometric methods for the quantitative
in vitro determination of DNA fragmentation are available. Examples
of such assays, including TUNEL (which detects incorporation of
labeled nucleotides in fragmented DNA) and ELISA-based assays, are
described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular
Biochemicals).
[0503] Apoptosis can also be determined by measuring morphological
changes in a cell. For example, as with necrosis, loss of plasma
membrane integrity can be determined by measuring uptake of certain
dyes (e.g., a fluorescent dye such as, for example, acridine orange
or ethidium bromide). A method for measuring apoptotic cell number
has been described by Duke and Cohen, Current Protocols in
Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). Cells
also can be labeled with a DNA dye (e.g., acridine orange, ethidium
bromide, or propidium iodide) and the cells observed for chromatin
condensation and margination along the inner nuclear membrane.
Other morphological changes that can be measured to determine
apoptosis include, e.g., cytoplasmic condensation, increased
membrane blebbing, and cellular shrinkage.
[0504] The presence of apoptotic cells can be measured in both the
attached and "floating" compartments of the cultures. For example,
both compartments can be collected by removing the supernatant,
trypsinizing the attached cells, combining the preparations
following a centrifugation wash step (e.g., 10 minutes at 2000
rpm), and detecting apoptosis (e.g., by measuring DNA
fragmentation). (See, e.g., Piazza et al., 1995, Cancer Research
55:3110-16).
[0505] The effects of Ligand Drug conjugates can be tested or
validated in animal models. A number of established animal models
of cancers are known to the skilled artisan, any of which can be
used to assay the efficacy of a Ligand Drug conjugate. Non-limiting
examples of such models are described infra. Moreover, small animal
models to examine the in vivo efficacies of Ligand Drug conjugates
can be created by implanting human tumor cell lines into
appropriate immunodeficient rodent strains, e.g., athymic nude mice
or SCID mice.
Ligand Unit
[0506] The Ligand unit (L) has at least one functional group that
can form a bond with a functional group of a Linker unit. Useful
functional groups that can be present on a Ligand unit, either
naturally, via chemical manipulation or via engineering, include,
but are not limited to, sulfhydryl (--SH), amino, hydroxyl,
carboxy, the anomeric hydroxyl group of a carbohydrate, and
carboxyl. In some embodiments, a Ligand unit functional group is a
sulfhydryl group. The sulfhydryl group is typically a solvent
accessible sulfhydryl group, such as a solvent accessible
sulfhydryl group on a cysteine residue. Sulfhydryl groups can be
generated by reduction of an intramolecular or intermolecular
disulfide bond of a Ligand. Sulfhydryl groups also can be generated
by reaction of an amino group of a lysine moiety of a Ligand using
2-iminothiolane (Traut's reagent) or another sulfhydryl generating
reagent.
[0507] In some embodiments, one or more sulfhydryl groups are
engineered into a Ligand unit, such as by amino acid substitution.
For example, a sulfhydryl group can be introduced into a Ligand
unit. In some embodiments, a sulfhydryl group is introduced by an
amino acid substitution of serine or threonine to a cysteine
residue, and/or by addition of a cysteine residue into a Ligand
unit (an engineered cysteine residue). In some embodiments, the
cysteine residue is an internal cysteine residue, i.e., not located
at the N-terminus or C-terminus of the Ligand moiety.
[0508] In an exemplary embodiment, a cysteine residue can be
engineered into an antibody heavy or light variable region (e.g.,
of an antibody fragment, such as a diabody) by amino acid
substitution. The amino acid substitution is typically introduced
into the framework region and is located distal to the
epitope-binding face of the variable region. For example, the amino
acid substitution can be at least 10 angstroms, at least 20
angstroms or at least 25 angstroms from the epitope-binding face or
the CDRs. Suitable positions for substitution of a cysteine residue
can be determined based on the known or predicted three dimensional
structures of antibody variable regions. (See generally Holliger
and Hudson, 2005, Nature BioTechnology 23(9):1126-1136.) In
exemplary embodiments, a serine to cysteine amino acid substitution
is introduced at amino acid position 84 of the V.sub.H region
and/or position 14 of the V.sub.L region (according to the
numbering system of Kabat et al., Sequences of Proteins of
Immunological Interest, 5th edition, (Bethesda, Md., NIH)
1991).
[0509] To control the number of Drug or Linker unit-Drug units
attached to a Ligand unit, one or more cysteine residues can be
eliminated by amino acid substitution. For example, the number of
solvent accessible cysteine residues in an immunoglobulin hinge
region can be reduced by amino acid substitution of cysteine to
serine residues.
[0510] In some embodiments, a Ligand unit contains 1, 2, 3, 4, 5, 6
7 or 8 solvent-accessible cysteine residues. In some embodiments, a
Ligand unit contains 2 or 4 solvent-accessible cysteine
residues.
Compositions and Methods of Administration
[0511] The CD19 binding agents and ligand-drug conjugate compounds
can be in any form that allows for the compound to be administered
to a patient for treatment of a CD19-associated disorder. For
example, the compound can be in the form of a liquid or solid.
Typical routes of administration include, without limitation,
parenteral, topical, oral, sublingual, rectal, vaginal, ocular,
intra-tumor, and intranasal. Parenteral administration includes
subcutaneous injections, intravenous, intramuscular, intrasternal
injection or infusion techniques. In one aspect, the compositions
are administered parenterally. In yet another aspect, the compounds
are administered intravenously.
[0512] Pharmaceutical compositions can be formulated so as to allow
a compound to be bioavailable upon administration of the
composition to a patient. Compositions can take the form of one or
more dosage units, where for example, a tablet can be a single
dosage unit.
[0513] Materials used in preparing the pharmaceutical compositions
can be non-toxic in the amounts used. It will be evident to those
of ordinary skill in the art that the optimal dosage of the active
ingredient(s) in the pharmaceutical composition will depend on a
variety of factors. Relevant factors include, without limitation,
the type of animal (e.g., human), the particular form of the
compound, the manner of administration, and the composition
employed.
[0514] The pharmaceutically acceptable carrier or vehicle can be
particulate, so that the compositions are, for example, in tablet
or powder form. The carrier(s) can be liquid, with the compositions
being, for example, an oral syrup or injectable liquid.
[0515] When intended for oral administration, the composition is
preferably in solid or liquid form, where semi-solid, semi-liquid,
suspension and gel forms are included within the forms considered
herein as either solid or liquid.
[0516] As a solid composition for oral administration, the
composition can be formulated into a powder, granule, compressed
tablet, pill, capsule, chewing gum, wafer or the like form. Such a
solid composition typically contains one or more inert diluents. In
addition, one or more of the following can be present: binders such
as carboxymethylcellulose, ethyl cellulose, microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or
dextrins, disintegrating agents such as alginic acid, sodium
alginate, Primogel, corn starch; lubricants such as magnesium
stearate or Sterotex; glidants such as colloidal silicon dioxide;
sweetening agents such as sucrose or saccharin, a flavoring agent
such as peppermint, methyl salicylate or orange flavoring, and a
coloring agent.
[0517] When the composition is in the form of a capsule, e.g., a
gelatin capsule, it can contain, in addition to materials of the
above type, a liquid carrier such as polyethylene glycol,
cyclodextrin or a fatty oil.
[0518] The composition can be in the form of a liquid, e.g., an
elixir, syrup, solution, emulsion or suspension. The liquid can be
useful for oral administration or for delivery by injection. When
intended for oral administration, a composition can comprise one or
more of a sweetening agent, preservatives, dye/colorant and flavor
enhancer. In a composition for administration by injection, one or
more of a surfactant, preservative, wetting agent, dispersing
agent, suspending agent, buffer, stabilizer and isotonic agent can
also be included.
[0519] The liquid compositions, whether they are solutions,
suspensions or other like form, can also include one or more of the
following: sterile diluents such as water for injection, saline
solution, preferably physiological saline, Ringer's solution,
isotonic sodium chloride, fixed oils such as synthetic mono or
digylcerides which can serve as the solvent or suspending medium,
polyethylene glycols, glycerin, cyclodextrin, propylene glycol or
other solvents; antibacterial agents such as benzyl alcohol or
methyl paraben; antioxidants such as ascorbic acid or sodium
bisulfate; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as amino acids, acetates, citrates or
phosphates; detergents, such as nonionic surfactants, polyols; and
agents for the adjustment of tonicity such as sodium chloride or
dextrose. A parenteral composition can be enclosed in ampoule, a
disposable syringe or a multiple-dose vial made of glass, plastic
or other material. Physiological saline is an exemplary adjuvant.
An injectable composition is preferably sterile.
[0520] The amount of the compound that is effective in the
treatment of a particular disorder or condition will depend on the
nature of the disorder or condition, and can be determined by
standard clinical techniques. In addition, in vitro or in vivo
assays can optionally be employed to help identify optimal dosage
ranges. The precise dose to be employed in the compositions will
also depend on the route of administration, and the seriousness of
the disease or disorder, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
[0521] The compositions comprise an effective amount of a compound
such that a suitable dosage will be obtained. Typically, this
amount is at least about 0.01% of a compound by weight of the
composition. When intended for oral administration, this amount can
be varied to range from about 0.1% to about 80% by weight of the
composition. In one aspect, oral compositions can comprise from
about 4% to about 50% of the compound by weight of the composition.
In yet another aspect, present compositions are prepared so that a
parenteral dosage unit contains from about 0.01% to about 2% by
weight of the compound.
[0522] For intravenous administration, the composition can comprise
from about 0.01 to about 100 mg of a compound per kg of the
animal's body weight. In one aspect, the composition can include
from about 1 to about 100 mg of a compound per kg of the animal's
body weight. In another aspect, the amount administered will be in
the range from about 0.1 to about 25 mg/kg of body weight of a
compound.
[0523] Generally, the dosage of a compound administered to a
patient is typically about 0.01 mg/kg to about 100 mg/kg of the
subject's body weight. In some embodiments, the dosage administered
to a patient is between about 0.01 mg/kg to about 15 mg/kg of the
subject's body weight. In some embodiments, the dosage administered
to a patient is between about 0.1 mg/kg and about 15 mg/kg of the
subject's body weight. In some embodiments, the dosage administered
to a patient is between about 0.1 mg/kg and about 20 mg/kg of the
subject's body weight. In some embodiments, the dosage administered
is between about 0.1 mg/kg to about 5 mg/kg or about 0.1 mg/kg to
about 10 mg/kg of the subject's body weight. In some embodiments,
the dosage administered is between about 1 mg/kg to about 15 mg/kg
of the subject's body weight. In some embodiments, the dosage
administered is between about 1 mg/kg to about 10 mg/kg of the
subject's body weight. In some embodiments, the dosage administered
is between about 0.1 to 4 mg/kg, even more preferably 0.1 to 3.2
mg/kg, or even more preferably 0.1 to 2.7 mg/kg of the subject's
body weight over a treatment cycle.
[0524] The compound or compositions can be administered by any
convenient route, for example by infusion or bolus injection, by
absorption through epithelial or mucocutaneous linings (e.g., oral
mucosa, rectal and intestinal mucosa). Administration can be
systemic or local. Various delivery systems are known, e.g.,
encapsulation in liposomes, microparticles, microcapsules,
capsules, and can be used to administer a compound. In certain
embodiments, more than one compounds or composition is administered
to a patient.
[0525] In specific embodiments, it can be desirable to administer
one or more compounds or compositions locally to the area in need
of treatment. This can be achieved, for example, and not by way of
limitation, by local infusion during surgery; topical application,
e.g., in conjunction with a wound dressing after surgery; by
injection; by means of a catheter; by means of a suppository; or by
means of an implant, the implant being of a porous, non-porous, or
gelatinous material, including membranes, such as sialastic
membranes, or fibers. In one embodiment, administration can be by
direct injection at the site (or former site) of a cancer, tumor or
neoplastic or pre-neoplastic tissue. In another embodiment,
administration can be by direct injection at the site (or former
site) of a manifestation of B cell disease such as, for example, a
malignancy and/or B-cell lineage malignancies.
[0526] In certain embodiments, it can be desirable to introduce one
or more compounds or compositions into the central nervous system
by any suitable route, including intraventricular and intrathecal
injection. Intraventricular injection can be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir.
[0527] In yet another embodiment, the compound or compositions can
be delivered in a controlled release system, such as but not
limited to, a pump or various polymeric materials can be used. In
yet another embodiment, a controlled-release system can be placed
in proximity of the target of the compound or compositions, e.g.,
the brain, thus requiring only a fraction of the systemic dose
(see, e.g., Goodson, in Medical Applications of Controlled Release,
supra, vol. 2, pp. 115-138 (1984)). Other controlled-release
systems discussed in the review by Langer (1990, Science
249:1527-1533) can be used.
[0528] The term "carrier" refers to a diluent, adjuvant or
excipient, with which a compound is administered. Such
pharmaceutical carriers can be liquids, such as water and oils,
including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil.
The carriers can be saline, gum acacia, gelatin, starch paste,
talc, keratin, colloidal silica, urea. In addition, auxiliary,
stabilizing, thickening, lubricating and coloring agents can be
used. In one embodiment, when administered to a patient, the
compound or compositions and pharmaceutically acceptable carriers
are sterile. Water is an exemplary carrier when the compounds are
administered intravenously. Saline solutions and aqueous dextrose
and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical
carriers also include excipients such as starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol. The present
compositions, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents.
[0529] The present compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, pellets, capsules, capsules
containing liquids, powders, sustained-release formulations,
suppositories, emulsions, suspensions, or any other form suitable
for use. Other examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin,
Mack Publishing Company, Easton, Pa., 18.sup.th Edition, 1995.
[0530] In an embodiment, the compounds are formulated in accordance
with routine procedures as a pharmaceutical composition adapted for
intravenous administration to animals, particularly human beings.
Typically, the carriers or vehicles for intravenous administration
are sterile isotonic aqueous buffer solutions. Where necessary, the
compositions can also include a solubilizing agent. Compositions
for intravenous administration can optionally comprise a local
anesthetic such as lignocaine to ease pain at the site of the
injection. Generally, the ingredients are supplied either
separately or mixed together in unit dosage form, for example, as a
dry lyophilized powder or water free concentrate in a hermetically
sealed container such as an ampoule or sachette indicating the
quantity of active agent. Where compound is to be administered by
infusion, it can be dispensed, for example, with an infusion bottle
containing sterile pharmaceutical grade water or saline. Where the
compound is administered by injection, an ampoule of sterile water
for injection or saline can be provided so that the ingredients can
be mixed prior to administration.
[0531] Compositions for oral delivery can be in the form of
tablets, lozenges, aqueous or oily suspensions, granules, powders,
emulsions, capsules, syrups, or elixirs, for example. Orally
administered compositions can contain one or more optionally
agents, for example, sweetening agents such as fructose, aspartame
or saccharin; flavoring agents such as peppermint, oil of
wintergreen, or cherry; coloring agents; and preserving agents, to
provide a pharmaceutically palatable preparation. Moreover, where
in tablet or pill form, the compositions can be coated to delay
disintegration and absorption in the gastrointestinal tract thereby
providing a sustained action over an extended period of time.
Selectively permeable membranes surrounding an osmotically active
driving compound are also suitable for orally administered
compounds. In these later platforms, fluid from the environment
surrounding the capsule is imbibed by the driving compound, which
swells to displace the agent or agent composition through an
aperture. These delivery platforms can provide an essentially zero
order delivery profile as opposed to the spiked profiles of
immediate release formulations. A time-delay material such as
glycerol monostearate or glycerol stearate can also be used.
[0532] The compositions can be intended for topical administration,
in which case the carrier can be in the form of a solution,
emulsion, ointment or gel base. If intended for transdermal
administration, the composition can be in the form of a transdermal
patch or an iontophoresis device. Topical formulations can comprise
a concentration of a compound of from about 0.05% to about 50% w/v
(weight per unit volume of composition), in another aspect, from
0.1% to 10% w/v.
[0533] The composition can include various materials that modify
the physical form of a solid or liquid dosage unit. For example,
the composition can include materials that form a coating shell
around the active ingredients. The materials that form the coating
shell are typically inert, and can be selected from, for example,
sugar, shellac, and other enteric coating agents. Alternatively,
the active ingredients can be encased in a gelatin capsule.
[0534] Whether in solid or liquid form, the present compositions
can include a pharmacological agent used in the treatment of
cancer, e.g., B-cell lineage malignancies.
[0535] The pharmaceutical compositions are generally formulated as
sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
CD19-Associated Disorders
[0536] The CD19 binding agents described herein, as well as
ligand-drug conjugate compounds, can be useful for treating or
preventing a CD19-expressing cancer or an immunological disorder
characterized by expression or overexpression of CD19. Such
expression of CD19 can be due to, for example, increased CD19
protein levels on the cell surface and/or altered antigenicity of
the expressed CD19. Treatment or prevention of the immunological
disorder, according to the methods described herein, is achieved by
administering to a subject in need of such treatment or prevention
an effective amount of the CD19 binding agent or ligand-drug
conjugate compound. In preferred embodiments, the ligand-drug
conjugate will (i) bind to activated immune cells that express CD19
and that are associated with the disease state and (ii) exert a
cytotoxic, cytostatic, or immunomodulatory effect on the activated
immune cells.
[0537] Immunological diseases that are characterized by
inappropriate activation of immune cells and that can be treated or
prevented by the methods described herein can be classified, for
example, by the type(s) of hypersensitivity reaction(s) that
underlie the disorder. These reactions are typically classified
into four types: anaphylactic reactions, cytotoxic (cytolytic)
reactions, immune complex reactions, or cell-mediated immunity
(CMI) reactions (also referred to as delayed-type hypersensitivity
(DTH) reactions). (See, e.g., Fundamental Immunology, William E.
Paul ed., Raven Press, N.Y., 3rd ed. 1993.)
[0538] Specific examples of such immunological diseases include,
but are not limited to, rheumatoid arthritis, multiple sclerosis,
endocrine ophthalmopathy, uveoretinitis, systemic lupus
erythematosus, myasthenia gravis, Grave's disease,
glomerulonephritis, autoimmune hepatological disorder, autoimmune
inflammatory bowel disease, anaphylaxis, allergic reaction,
Sjogren's syndrome, juvenile onset (Type I) diabetes mellitus,
primary biliary cirrhosis, Wegener's granulomatosis, fibromyalgia,
inflammatory bowel disease, polymyositis, dermatomyositis, multiple
endocrine failure, Schmidt's syndrome, autoimmune uveitis,
Addison's disease, adrenalitis, thyroiditis, Hashimoto's
thyroiditis, autoimmune thyroid disease, pernicious anemia, gastric
atrophy, chronic hepatitis, lupoid hepatitis, atherosclerosis,
presenile dementia, demyelinating diseases, subacute cutaneous
lupus erythematosus, hypoparathyroidism, Dressler's syndrome,
autoimmune thrombocytopenia, idiopathic thrombocytopenic purpura,
hemolytic anemia, pemphigus vulgaris, pemphigus, dermatitis
herpetiformis, alopecia arcata, pemphigoid, scleroderma,
progressive systemic sclerosis, CREST syndrome (calcinosis,
Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and
telangiectasia), adult onset diabetes mellitus (Type II diabetes),
male and female autoimmune infertility, ankylosing spondolytis,
ulcerative colitis, Crohn's disease, mixed connective tissue
disease, polyarteritis nedosa, systemic necrotizing vasculitis,
juvenile onset rheumatoid arthritis, atopic dermatitis, atopic
rhinitis, Goodpasture's syndrome, Chagas' disease, sarcoidosis,
rheumatic fever, asthma, recurrent abortion, anti-phospholipid
syndrome, farmer's lung, erythema multiforme, post cardiotomy
syndrome, Cushing's syndrome, autoimmune chronic active hepatitis,
bird-fancier's lung, allergic encephalomyelitis, toxic epidermal
necrolysis, Alport's syndrome, alveolitis, allergic alveolitis,
fibrosing alveolitis, interstitial lung disease, erythema nodosum,
pyoderma gangrenosum, transfusion reaction, leprosy, malaria,
leishmaniasis, trypanosomiasis, Takayasu's arteritis, polymyalgia
rheumatica, temporal arteritis, schistosomiasis, giant cell
arteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,
lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,
Kawasaki's disease, dengue, encephalomyelitis, endocarditis,
endomyocardial fibrosis, endophthalmitis, erythema elevatum et
diutinum, psoriasis, erythroblastosis fetalis, eosinophilic
faciitis, Shulman's syndrome, Felty's syndrome, filariasis,
cyclitis, chronic cyclitis, heterochronic cyclitis, Fuch's
cyclitis, IgA nephropathy, Henoch-Schonlein purpura, graft versus
host disease, transplantation rejection, human immunodeficiency
virus infection, echovirus infection, cardiomyopathy, Alzheimer's
disease, parvovirus infection, rubella virus infection, post
vaccination syndromes, congenital rubella infection, Eaton-Lambert
syndrome, relapsing polychondritis, cryoglobulinemia, Waldenstrom's
macroglobulemia, Epstein-Barr virus infection, mumps, Evan's
syndrome, and autoimmune gonadal failure.
[0539] Accordingly, the methods described herein encompass
treatment of disorders of B lymphocytes (e.g., systemic lupus
erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and
type I diabetes), Th.sub.1-lymphocytes (e.g., rheumatoid arthritis,
multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's
thyroiditis, Grave's disease, primary biliary cirrhosis, Wegener's
granulomatosis, tuberculosis, or graft versus host disease), or
Th.sub.2-lymphocytes (e.g., atopic dermatitis, systemic lupus
erythematosus, atopic asthma, rhinoconjunctivitis, allergic
rhinitis, or chronic graft versus host disease). Generally,
disorders involving dendritic cells involve disorders of
Th.sub.1-lymphocytes or Th.sub.2-lymphocytes.
[0540] The invention also includes treatment of an autoimmune
disease, for example an autoimmune disease that is mediated at
least in part by B cells. Examples of autoimmune diseases include
acute necrotizing hemorrhagic leukoencephalitis; Addison's disease;
Agammaglobulinemia; Allergic asthma; Allergic rhinitis; Alopecia
areata; Amyloidosis; Ankylosing spondylitis; Anti-GBM/Anti-TBM
nephritis; Antiphospholipid syndrome; Autoimmune aplastic anemia;
Autoimmune dysautonomia; Autoimmune hepatitis; Autoimmune
hyperlipidemia; Autoimmune immunodeficiency; Autoimmune inner ear
disease; Autoimmune myocarditis; Autoimmune thrombocytopenic
purpura; Axonal & neuronal neuropathies; Balo disease; Behcet's
disease; Bullous pemphigoid; Cardiomyopathy; Castleman disease;
Celiac sprue (nontropical); Chagas disease; Chronic fatigue
syndrome; Chronic inflammatory demyelinating polyneuropathy;
Churg-Strauss syndrome; Cicatricial pemphigoid/benign mucosal
pemphigoid; Crohn's disease; Cogans syndrome; Cold agglutinin
disease; Congenital heart block; Coxsackie myocarditis; CREST
disease; Essential mixed cryoglobulinemia; Demyelinating
neuropathies; Dermatomyositis; Devic disease; Discoid lupus;
Dressler's syndrome; Endometriosis; Eosinophilic fasciitis;
Erythema nodosum; Experimental allergic encephalomyelitis; Evans
syndrome; Fibromyalgia; Fibrosing alveolitis; Giant cell arteritis
(temporal arteritis); Goodpasture's syndrome; Graves' disease;
Guillain-Barre syndrome; Hashimoto's disease; Hemolytic anemia;
Henoch-Schonlein purpura; Herpes gestationis;
Hypogammaglobulinemia; Idiopathic thrombocytopenic purpura; IgA
nephropathy; Immunoregulatory lipoproteins; Inclusion body
myositis; Insulin-dependent diabetes (type1); Interstitial
cystitis; Juvenile arthritis; Juvenile diabetes; Kawasaki syndrome;
Lambert-Eaton syndrome; Leukocytoclastic vasculitis; Lichen planus;
Lichen sclerosus; Ligneous conjunctivitis; Linear IgA disease
(LAD); Lupus (SLE); Lyme disease; Meniere's disease; Microscopic
polyangiitis; Mixed connective tissue disease; Mooren's ulcer;
Mucha-Habermann disease; Multiple sclerosis; Myasthenia gravis;
Myositis; Narcolepsy; Neutropenia; Ocular cicatricial pemphigoid;
Osteoarthritis; Palindromic rheumatism; Paraneoplastic cerebellar
degeneration; Paroxysmal nocturnal hemoglobinuria;
Parsonnage-Turner syndrome; Pars planitis (peripheral uveitis);
Pemphigus; Peripheral neuropathy; Perivenous encephalomyelitis;
Pernicious anemia; POEMS syndrome; Polyarteritis nodosa; Type I,
II, & III autoimmune polyglandular syndromes; Polymyalgia
rheumatica; Polymyositis; Postmyocardial infarction syndrome;
Postpericardiotomy syndrome; Progesterone dermatitis; Primary
biliary cirrhosis; Psoriasis; Psoriatic arthritis; Idiopathic
pulmonary fibrosis; Pyoderma gangrenosum; Pure red cell aplasia;
Raynauds phenomenon; Reflex sympathetic dystrophy; Reiter's
syndrome; Relapsing polychondritis; Restless legs syndrome;
Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Schmidt
syndrome; Scleritis; Scleroderma; Sjogren's syndrome; Sperm &
testicular autoimmunity; Stiff person syndrome; Subacute bacterial
endocarditis; Sympathetic ophthalmia; Takayasu's arteritis;
Temporal arteritis/Giant cell arteritis; Thrombocytopenic purpura;
Autoimmune thyroid disease; Tolosa-Hunt syndrome; Transverse
myelitis & necrotizing myelopathy; Ulcerative colitis;
Undifferentiated connective tissue disease; Uveitis; Vasculitis;
Vesiculobullous dermatosis; Vitiligo; and Wegener's granulomatosis.
The more common autoimmune diseases that are of especial interest
include (a) connective tissue diseases such as systemic lupus
erythematosus, rheumatoid arthritis, systemic sclerosos
(scleroderma), Sjogren's syndrome, (b) neuromuscular diseases such
as multiple sclerosos, myasthenis gravis, Guillain-Barre syndrome,
(c) endocrine diseases such as Hashimoto's thryoiditis, Grave's
disease, insulin-dependent (type 1) diabetes, and (d)
gastrointestinal diseases such as inflammatory bowel disease
(including Crohn's disease and ulcerative colitis), and (e) other
diseases such as vasculitis syndromes, hematologic autoimmune
diseases, and autoimmune skin diseases.
[0541] The autoimmune disease for example includes the presence of
autoantibodies. The autoantibody can bind specifically to host
targets or antigens, for example rheumatoid factor (e.g., in
rheumatoid arthritis); topoisomerase (e.g., in scleroderma); myelin
basic protein (e.g., in multiple sclerosis); basement membrane
collagen type iv protein (e.g., in Goodpasture's syndrome);
ganglioside (e.g., in Guillain-Barre syndrome); platelets (e.g.,
chronic idiopathic thrombocytopenia); smooth muscle actin (e.g., in
autoimmune hepatitis); bullous pemphigoid antigen 1 and 2; also
called hemidesmosome antigens (e.g., in bullous pemphigoid);
transglutaminase (e.g., in coeliac disease); desmogein 3 (e.g., in
pemphigus vulgaris); p62 or sp100 or mitochondrial (m2) antigens
(e.g., in primary biliary cirrhosis); neutrophil cytoplasmic c-ANCA
(e.g., in Wegener's granulomatosis); neutrophil perinuclear p-ANCA
(e.g., Polyarteritis nodosa, Microscopic polyangiitis,
Churg-Strauss syndrome, Systemic vasculitides (non-specific));
double-stranded-DNA (e.g., in systemic lupus erythematosus);
exosome complex (e.g., in Scleromyositis); Ro or La antigen (e.g.,
in systemic lupus erythematosus and neonatal heart block, or
primary Sjogren's syndrome); Smith antigen (e.g., in systemic lupus
erythematosus); phospholipid antigen (e.g., in antiphospholipid
syndrome); SSA or SSB antigen (e.g., in Sjogren's syndrome);
centromere (e.g., in CREST syndrome; mitochondria (e.g., in primary
biliary cirrhosis); nicotinic acetylcholine receptor (e.g., in
myasthenia gravis); voltage-gated calcium channel (e.g., in
Lambert-Eaton syndrome); thyroid peroxidase (e.g., in Hashimoto's
thyroiditis); TSH receptor (e.g., in Graves' disease); Hu antigen
(e.g., in paraneoplastic cerebellar syndrome); voltage-gated
potassium channel (e.g., in limbic encephalitis and
N-methyl-D-aspartate receptor (e.g., in encephalitis). More than
one type of autoantibody can be associated with an immunological
disorder or visa versa, and this list in not exhaustive. For
example, autoantigens that have been identified in rheumatoid
arthritis include joint-associated proteins such as collagen type
II, human chondrocyte glycoprotein 39, and proteoglycans; as well
as heat shock proteins, citrullinated filaggrin, immunoglobulin,
glucose-6-phosphate isomerase, p205, and BiP.
[0542] The CD19-binding agent can be administered in an amount
effective to mitigate at least one symptom of the autoimmune
disorder. The invention provides treatment of an autoimmune disease
that is refractory to conventional therapy with at least one
CD19-binding agent. The CD19 binding agent is optionally
administered in combination with another therapy, e.g., surgery,
anti-inflammatory drug therapy, hormone/enzyme replacement therapy,
plasmapheresis and immunosuppressant therapy. Anti-inflammatory
drug therapies include steroids, e.g., corticosteroids such as
prednisone; as well as NSAIDs such as salicylates and other COX
inhibitors. Hormone replacement therapy includes thyroid hormone
replacement (e.g., in Hashimoto's Thryoiditis). Immunosuppressant
drugs include glucocorticoids, alkylating agents (e.g.,
cyclophosphamide, often effective in SLE), and antimetabolites
(e.g., methotrexate, azathioprine and mercaptopurine). Other
therapies include antithyroid drug therapy or removal of the
thyroid gland surgically or by radioiodine (e.g., in Grave's
disease).
[0543] The CD19 binding agents, as well as ligand-drug conjugate
compounds, are also useful for treating or preventing a
CD19-expressing cancer. Treatment or prevention of a
CD19-expressing cancer, according to the methods described herein,
is achieved by administering to a subject in need of such treatment
or prevention an effective amount of the CD19 binding agent, or
ligand-drug conjugate compound whereby the agent or compound (i)
binds to CD19-expressing cancer cells and (ii) exerts a cytotoxic
or cytostatic effect to, for example, deplete or inhibit the
proliferation of the CD19-expressing cancer cells.
[0544] Cancers that can be treated or prevented by the methods
described herein include, for example, B cell malignancies,
including, for example, B cell subtype non-Hodgkin's lymphoma (NHL)
including low grade/follicular NHL, small lymphocytic (SL) NHL,
intermediate grade/follicular NHL, intermediate grade diffuse NHL,
diffuse large B-cell lymphoma, follicular lymphoma, high grade
immunoblastic NHL, high grade lymphoblastic NHL, high grade small
non-cleaved cell NHL and bulky disease NHL; Burkitt's lymphoma;
multiple myeloma; pre-B acute lymphoblastic leukemia and other
malignancies that derive from early B cell precursors; common acute
lymphoblastic leukemia; chronic lymphocytic leukemia; hairy cell
leukemia; Null-acute lymphoblastic leukemia; Waldenstrom's
Macroglobulinemia; and pro-lymphocytic leukemia; light chain
disease; plasmacytoma; osteosclerotic myeloma; plasma cell
leukemia; monoclonal gammopathy of undetermined significance
(MGUS); smoldering multiple myeloma (SMM); indolent multiple
myeloma (IMM); or Hodgkin's lymphoma, provided that the cancers
express the CD19 antigen.
Therapeutic Uses of the Compounds of the Present Invention
[0545] In therapeutic applications, at least one CD19 binding agent
(e.g., an antibody or an antibody-drug conjugate) is administered
to a patient suspected of, or already known to be suffering from, a
CD19-associated disorder, e.g., a cancer. The agent for example is
administered in an amount sufficient to abolish, or at least
lessen, at least one symptom of the disorder.
[0546] In prophylactic applications of treatment, at least one
agent is administered to a patient at risk of developing or
suffering a relapse of a CD19-associated disorder. The patient is
for example a patient in apparent remission of a CD19-associated
disorder, for whom there is a possibility of a relapse, or a
patient who is at enhanced risk of an increase in at least one
symptom of a CD19-associated disorder relative to the general
population. Patients known to be at high risk of a CD19-associated
disorder or its relapse include, e.g., patients diagnosed with an
aggressive form of the disorder, or with genetic or histological
abnormalities associated with the disorder or its staging (e.g.,
malignancy), or with an associated risk factor (e.g., familial
history or another CD19 associated disorder or EBV infection).
[0547] The agent can be administered before a suspected onset or
increase or exacerbation or relapse of the disorder, in an amount
sufficient to eliminate, or reduce the risk of, or delay the onset
or relapse of the disorder.
[0548] The CD19 binding agents and ligand-drug conjugate compounds
are useful for treating cancer and other diseases in which CD19 is
expressed or overexpressed, relative to normal (e.g., non-cancerous
tissue). The CD19 binding agents can also be used to treat
CD19-associated disorders in which CD19 is not overexpressed
relative to normal. For example, the disorder can include an
increased count of CD19-positive B cells. In some embodiments, the
CD19 binding agents and ligand-drug conjugate compounds are
administered alone. In other embodiments, the CD19 binding agents
and the ligand-drug conjugate compounds are coadministered with
another therapeutic agent, or administered sequentially. In some
embodiments, the CD19 binding agents and ligand-drug conjugate
compounds coadministered with standard of care chemotherapeutics,
or administered sequentially.
[0549] The response of the patient can be monitored by determining
the effect of the agent on a CD19-associated disorder.
Treatment of Cancer
[0550] The CD19 binding agents and ligand-drug conjugate compounds
are useful for inhibiting the multiplication of a tumor cell or
cancer cell, causing apoptosis in a tumor or cancer cell, or for
treating cancer in a patient. The compounds can be used accordingly
in a variety of settings for the treatment of cancers. The
conjugate compounds provide antigen-specific tumor or cancer
targeting, thus reducing potential toxicity of the Drug.
[0551] In some embodiments, the cancer (e.g., B cell lymphoma and a
leukemia) is a cancer expressing or over-expressing CD19 (i.e.,
relative to normal tissue), such as in B-lymphocytes.
Multi-Modality Therapy for Cancer
[0552] Cancers, including, but not limited to, a tumor, metastasis,
or other disease or disorder characterized by uncontrolled cell
growth, can be treated or prevented by administration of compounds
according to the present invention, i.e., CD19 binding agents or
ligand-drug conjugate compounds.
[0553] In some embodiments, methods for treating or preventing
cancer are provided, including administering to a patient in need
thereof an effective amount of a compound of the present invention
and a chemotherapeutic agent. In one embodiment, the
chemotherapeutic agent is that with which treatment of the cancer
has not been found to be refractory. In another embodiment, the
chemotherapeutic agent is that with which the treatment of cancer
has been found to be refractory. The compounds of the present
invention can be administered to a patient that has also undergone
surgery as treatment for the cancer.
[0554] In one embodiment, the additional treatment is radiation
therapy.
[0555] In a specific embodiment, the CD19 binding agent or CD19
ligand-drug conjugate compound is administered concurrently or
sequentially with the chemotherapeutic agent and/or with radiation
therapy. In another specific embodiment, the chemotherapeutic agent
or radiation therapy is administered prior or subsequent to
administration of the CD19 binding agent, or anti-CD19 ligand-drug
conjugate compound. In some embodiments, the chemotherapeutic agent
or radiation therapy is administered at least an hour, five hours,
12 hours, a day, a week, a month, several months (e.g., up to three
months), prior or subsequent to administration of a compound of the
present invention.
[0556] Where a compound of the present invention and
chemotherapeutic drug(s) are administered separately, the number of
dosages of each compound given per day, may not necessarily be the
same, e.g. where one compound may have a greater duration of
activity, and will therefore, be administered less frequently. The
compound of the present invention and the chemotherapeutic drug(s)
can be administered via the same or different routes of
administration. They can be administered according to simultaneous
or alternating regimens, at the same or different times during the
course of the therapy, concurrently in divided or single forms.
Administration of either or both agents can be on a continous
basis, e.g., by infusion or via an implanted reservoir.
[0557] A chemotherapeutic agent can be administered over a series
of sessions. Any one or a combination of the following
chemotherapeutic agents can be administered (see infra). With
respect to radiation, any radiation therapy protocol can be used
depending upon the type of cancer to be treated. For example, but
not by way of limitation, x-ray radiation can be administered; in
particular, high-energy megavoltage (radiation of greater that 1
MeV energy) can be used for deep tumors, and electron beam and
orthovoltage x-ray radiation can be used. Gamma-ray emitting
radioisotopes, such as radioactive isotopes of radium, cobalt and
other elements, can also be administered.
[0558] Additionally, methods of treatment of cancer with a compound
of the present invention are provided as an alternative to
chemotherapy or radiation therapy where the chemotherapy or the
radiation therapy has proven or can prove too toxic, e.g., results
in unacceptable or unbearable side effects, for the subject being
treated. The subject being treated can, optionally, be treated with
another cancer treatment such as surgery, radiation therapy or
chemotherapy, depending on which treatment is found to be
acceptable or bearable.
[0559] Compounds of the present invention can also be used in an in
vitro or ex vivo fashion, such as for the treatment of certain
cancers.
Multi-Drug Therapy for Cancer
[0560] Methods for treating cancer including administering to a
patient in need thereof an effective amount of an CD19 binding
agent or ligand-drug conjugate compound and another therapeutic
agent that is an anti-cancer agent.
[0561] In some embodiments, the other therapeutic agent will be an
agent that is standard of care for the specific disease to be
treated or is part of a salvage regimen for the specific disease to
be treated. Anti-cancer agents and chemotherapeutic regimens
include, for example, anti-cancer antibodies, including, for
example, anti-CD52 antibodies (e.g., Alemtuzumab), anti-CD20
antibodies (e.g., Rituximab), and anti-CD40 antibodies (e.g.,
SGN40); chemotherapeutic regimens including, for example, CHOP
(cyclophosphamide, doxorubicin, vincristine, and prednisone); CVP
(cyclophosphamide, vincristine, and prednisone); RCVP
(Rituximab+CVP); RCHOP (Rituximab+CHOP); RICE (Rituximab+ifosamide,
carboplatin, etoposide); RDHAP, (Rituximab+dexamethasone,
cytarabine, cisplatin); RESHAP (Rituximab+etoposide,
methylprednisolone, cytarabine, cisplatin); gemcitabine;
combination treatment with vincristine, prednisone, and
anthracycline, with or without asparaginase; combination treatment
with daunorubicin, vincristine, prednisone, and asparaginase;
combination treatment with teniposide and Ara-C (cytarabine);
combination treatment with methotrexate and leucovorin; combination
treatment with bleomycin, doxorubicin, etoposide, mechlorethamine,
prednisone, vinblastine, and vincristine; small molecule
inhibitors; and proteosome inhibitors including, for example,
bortezomib.
[0562] The present invention encompasses methods of treating
lymphomas using the described CD19 binding agents or ligand-drug
conjugate compounds alone or in combination therapy with, for
example, anti-lymphoma antibodies, including, for example,
anti-CD20 antibodies, i.e., Rituximab, and/or anti-CD40 antibodies,
i.e., SGN-40.
[0563] The present invention encompasses methods of treating
lymphomas using the described CD19 binding agents or ligand-drug
conjugate compounds alone or in combination therapy with, for
example, chemotherapeutic regimens for the treatment of lymphomas
including, for example, CHOP (cyclophosphamide, doxorubicin,
vincristine, and prednisone), CVP (cyclophosphamide, vincristine,
and prednisone) and/or other anthracycline B chemotherapy
regimens.
[0564] The present invention encompasses methods of treating
indolent lymphomas using the described CD19 binding agents or
ligand-drug conjugate compounds alone or in combination therapy
with, for example, RCVP (Rituximab+CVP) and/or RCHOP
(Rituximab+CHOP).
[0565] The present invention encompasses methods of treating
subjects suffering from relapsed or refractory lymphoma using the
described CD19 binding agents or ligand-drug conjugate compounds
alone or in combination therapy with, for example, RICE
(Rituximab+ifosamide, carboplatin, etoposide), RDHAP,
(Rituximab+dexamethasone, cytarabine, cisplatin), RESHAP
(Rituximab+etoposide, methylprednisolone, cytarabine, cisplatin),
gemcitabine and/or an immune modulatory drugs, i.e.,
lenalidomide.
[0566] The present invention encompasses methods of treating a
subject that has relapsed disease or that is refractory to
treatment with Rituximab or other therapy for the treatment of
cancer, e.g., CHOP, CVP, CHOP, RICE, RDHAP, RCHOP, RCVP, RESHAP. In
one aspect, the methods include, for example, administering a
ligand-drug conjugate of the present invention to the subject. In
certain embodiments, the ligand-drug conjugate comprises a CD19
binding agent conjugated to an auristatin compound. In one aspect,
the CD19 binding agent is a humanized BU12 antibody.
[0567] The present invention encompasses methods of treating a
subject that has a cancer characterized by the level of CD21
expression. The cancer can have no, low levels, or high levels of
CD21 expression. In one aspect, the methods include, for example,
administering a ligand-drug conjugate of the present invention to
the subject. In certain embodiments, the ligand-drug conjugate
comprises a CD19 binding agent conjugated to an auristatin
compound. In one aspect, the CD19 binding agent is a humanized BU12
antibody.
[0568] The present invention encompasses methods of treating ALL
using the described CD19 binding agents or ligand-drug conjugate
compounds alone or in combination therapy with, for example, a
chemotherapeutic regimen that includes the combination of
vincristine, prednisone, and anthracycline, with or without
asparaginase. Alternative chemotherapeutic regimens include, for
example, combinations of daunorubicin, vincristine, prednisone, and
asparaginase; combinations of teniposide and ara-C(cytarabine);
combinations of methotrexate and leucovorin; combinations of
bleomycin, doxorubicin, etoposide, mechlorethamine, prednisone,
vinblastine and vincristine ("Stanford 5 Regimen").
[0569] Also within the scope of the invention are kits comprising
an isolated CD19 binding agent that specifically binds to human
CD19 or a ligand-drug conjugate comprising a CD19 binding agent,
and instructions for use. The kit can further contain a least one
additional reagent. Kits typically include a label indicating the
intended use of the contents of the kit. The term label includes
any writing, or recorded material supplied on or with the kit, or
which otherwise accompanies the kit.
[0570] The invention also includes diagnostic use of a humanized
CD19 antibody. For example, the humanized CD19 antibody can be used
as a diagnostic imaging agent alone and/or in combination with
other diagnostic imaging agents and/or in conjunction with
therapeutic applications. The diagnostic agent can be used in vivo
in human patients known to have or have had a CD19-associated
disorder. Optionally, the disorder includes CD19-positive cells
that are discrete localized, e.g., in a solid tumor.
[0571] In one such method, the CD19 binding agent can be directly
or indirectly labeled with a detectable label, such as a
fluorophore, and optionally contacted with a target cell or a
patient sample in vitro or in vivo. The presence and/or density of
CD 19 in a sample or individual can be determined. In vivo methods
of determination can include imaging techniques such as PET
(positron emission tomography) or SPECT (single photon emission
computed tomography).
[0572] The patient or sample (an optionally a control as well) is
for example contacted with a CD19 binding agent under conditions
that allow for formation of a complex between the agent and CD19
antigen if present. Complex formation is then detected in the test
patient and compared to binding in a control (e g, using an FACS
analysis or Western blotting). Any statistically significant
difference in the formation of complexes between the control and
the test sample/patient is indicative of the presence of a
CD19-associated disorder. The control can be e.g., a similar
reading taken from the same patient at a different location or
timepoint or a reading taken from non-diseased subjects, or a
predetermined statistical value based on multiple readings taken
from individuals selected at random or not known to be suffering
from the disorder. The diagnostic tests can be used to identify
patients with a CD19-associated disorder, or to determine the
extent of such a disorder in a particular patient, or to monitor
the course of a disorder over time, or the effect of a chosen
treatment on a disorder.
[0573] All publications and patent documents cited above are hereby
incorporated by reference in their entirety for all purposes to the
same extent as if each were so individually denoted.
[0574] The invention will be further described with reference to
the following examples; however, it is to be understood that the
invention is not limited to such examples.
[0575] The following examples of specific aspects for carrying out
the present invention are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention in
any way.
EXAMPLES
Example 1
Design of Humanized BU12 Heavy Chain Variable Region:
[0576] BU12 V.sub.H was aligned to functional human germline
V.sub.H exons. Selection of the V.sub.H exon was made based on
framework homology and canonical structure. Human germline V.sub.H
exons V.sub.H2-70 and V.sub.H4-31 were selected to provide
frameworks for humanization. Human germline J.sub.H4 exon was
selected to provide humanized FR4 sequence based on its identity
(85%) with FR4 of BU12 V.sub.H.
[0577] BU12 V.sub.H was aligned against the mouse V.sub.H exon
germline sequences to identify regions of somatic mutation which
may have structural implications. BU12 V.sub.H was found to have
high homology to the functional CB17H-10 V.sub.H exon with
potential framework regions of somatic mutation at H75, H82A and
H89.
[0578] BU12 V.sub.H was aligned against the selected human germline
V.sub.H exon (V.sub.H2-70 or V.sub.H4-31) and differences between
BU12 V.sub.H and the human framework at residues described in the
literature to effect CDR structure or V.sub.H/V.sub.L interactions
were identified. For the V.sub.H4-31 such residues changes were
found at positions H24, H27, H29 and H71. Additionally
non-homologous framework regions were identified and the crystal
structure of a homologous V.sub.H domain (1ETZ) was used to
determine the positions of non-homologous residues and assess their
likely impact on CDR structure.
Humanizing Mutations in Heavy Chain Variants
TABLE-US-00005 [0579] V.sub.H V.sub.H Exon Donor Variant Acceptor
Sequence Framework Residues V.sub.HA VH2-70 None V.sub.HB VH2-70
H75 V.sub.HC VH2-70 H79 V.sub.HD VH2-70 H81, H82, H82A, H82B, H82C
V.sub.HE VH2-70 H89 V.sub.HF VH4-31 None V.sub.HG VH4-31 H71
V.sub.HH VH4-31 H24, H27, H29 V.sub.HI VH4-31 H24, H27, H29, H71
V.sub.HJ VH4-31 H75 V.sub.HK VH4-31 H78, H79 V.sub.HL VH4-31
H89
TABLE-US-00006 Non-homologous FR Residues BU12 V.sub.H vs.
V.sub.H2-70 Position Change Comments H41 S .fwdarw. P Loop
region--exclude H75 S .fwdarw. K Possible somatic mutation/ charge
change H79 F .fwdarw. P Aromatic in core H81 K .fwdarw. T Core
(continuous region) H82 I .fwdarw. M Core (continuous region) H82A
A .fwdarw. T Core (continuous region) H82B S .fwdarw. N Core
(continuous region) H82C V .fwdarw. M Core (continuous region) H84
T .fwdarw. P Loop region--exclude H89 A .fwdarw. T Possible somatic
mutation
Specific Mutations in BU12 Heavy Chain Variants
TABLE-US-00007 [0580] Variant H75 H79 H81 H82 H82A H82B H82C H89
cBU12 VH S* F* K* I* A* S* V* A* VH2-70 K V T M T N M T HA K V T M
T N M T HB S* V T M T N M T HC K F* T M T N M T HD K V K* I* A* S*
V* T HE K V T M T N M A* *Mouse residues
TABLE-US-00008 Non-homologous FR Residues BU12 VH vs. VH4-31
Position Change Comments H3 T .fwdarw. Q Surface accessible distant
from CDRs--exclude H24 F .fwdarw. V Impacts CDR1 structure H27 F
.fwdarw. G Impacts CDR1 structure H29 L .fwdarw. I Impacts CDR1
structure H41 S .fwdarw. P Loop region--exclude H71 K .fwdarw. V
Impacts CDR2 structure H75 S .fwdarw. K Possible somatic
mutation/charge change H78 V .fwdarw. F Aromatic in core H79 F
.fwdarw. P Aromatic in core H83 D .fwdarw. T Loop region--exclude
H89 A .fwdarw. V Possible somatic mutation
Specific Mutations in BU12 Heavy Chain Variants
TABLE-US-00009 [0581] Variant H24 H27 H29 H71 H75 H78 H79 H89 cBU12
VH* F* F* L* K* S* V* F* A* VH4-31 V G I V K F S V HF V G I V K F S
V HG V G I K* K F S V HH F* F* L* V K F S V HI F* F* L* K* K F S V
HJ V G I V S* F S V HK V G I V K V* F* V HL V G I V K F S A* *Mouse
residues
Example 2
Design of Humanized BU12 Light Chain Variable Region:
[0582] BU12 V.sub.L was aligned to functional human germline
V.sub.H exons. Usage of L6 is high (.about.11%) so this was chosen
as the best framework for BU12 V.sub.L humanization. A10, with the
best homology to BU12 V.sub.L was also chosen. Human germline
J.sub..kappa.2 exon was selected to provide humanized FR4 sequence
based on its identity (77%) to FR4 of BU12 V.sub.L.
[0583] BU12 V.sub.L was aligned against the mouse V.sub.L exon
germline sequences to identify regions of somatic mutation which
may have structural implications. The closest matches were ac4, kn4
and kk4. Potential sites of somatic mutation were identified at
positions L40, L41, L42, L69, L71, L72 and L83.
[0584] BU12 V.sub.L was aligned against the selected human germline
and V.sub.L exon (L6 or A10) and differences between BU12 V.sub.L
and the human framework at residues described in the literature to
effect CDR structure or V.sub.H/V.sub.L interactions were
identified. Such residue differences occur at positions L2 and L71.
Additionally non-homologous framework regions were identified and
the crystal structure of a homologous V.sub.L domain (1QOK) was
used to determine the positions of non-homologous residues and
assess their likely impact on CDR structure.
Humanizing Mutations in Light Chain Variants
TABLE-US-00010 [0585] V.sub.L V.sub.H Exon Donor Variant Acceptor
Sequence Framework Residues V.sub.LA VL-L6 None V.sub.LB VL-L6 L2
V.sub.LC VL-L6 L71 V.sub.LD VL-L6 L2, L71 V.sub.LE VL-L6 L40, L41,
L42 V.sub.LF VL-L6 L69, L70, L71, L72 V.sub.LG VL-L6 L83 V.sub.LH
VL A10 None V.sub.LI VL A10 L2, L71
TABLE-US-00011 Non-homologous FR Residues BU12 VL vs. L6 and A10
Position Change Comments L2 N .fwdarw. I L2 known to impact CDR1
structure L40 S .fwdarw. P Possible somatic mutation L41 S .fwdarw.
G Possible somatic mutation L42 T .fwdarw. Q Possible somatic
mutation L69 N .fwdarw. T Possible somatic mutation L70 S .fwdarw.
D Charge in strand packing against CDR1 L71 H .fwdarw. F Somatic
mutation/L71 known to impact CDR1 structure L72 F .fwdarw. T
Possible somatic mutation L83 V .fwdarw. F Possible somatic
mutation
TABLE-US-00012 Specific mutations in BU12 1ight chain variants:
Variant L2 L40 L41 L42 L69 L70 L71 L72 L83 cBU12 VL* N* S* S* T* N*
S* H* F* V* L6 I P G Q T D F T F LA I P G Q T D F T F LB N* P G Q T
D F T F LC I P G Q T D H* T F LD N* P G Q T D H* T F LE I S* S* T*
T D F T F LF I P G Q N* S* H* F* F LG I P G Q T D F T V* *Mouse
residues
Specific Mutations in BU12 Light Chain Variants
TABLE-US-00013 [0586] Variant L2 L71 cBU12* N* H* A10 I F LH I F LI
N* H* *Mouse residues
Example 3
[0587] A panel of anti-CD19 antibodies was screened on a panel of
CD19+ NHL cell lines (FIG. 15). All of the antibodies evaluated
were able to deliver drug although there were differences between
the cell lines.
TABLE-US-00014 IC.sub.50 (ng/mL) of various anti-CD19 Antibodies
CD19 linked with 2.degree.-ADC Cell Line Disease Type
Molecules/Cell LT19 HIB19 cBU12 SJ25-C1 B-C3 CA46 Burkitt's
Lymphoma, 60527 4 1 7 4 17 EBV- HS Sultan Burkitt's Lymphoma, 59669
112 97 100 150 234 EBV+ HT Diffuse Mixed 35813 111 ~1000 238 ND ND
Lymphoma MC 116 Undifferentiated 29210 192 188 186 ~200 195
Lymphoma Ramos Burkitt's Lymphoma, 34377 1 1 5 3 13 EBV- Toledo
Diffuse Large Cell 28657 584 ~1000 ~1000 512 359 Lymphoma
[0588] Anti-CD19 antibodies deliver
2.degree.-goat-anti-mouse-vcMMAF to CD19.sup.+ cell lines. Cell
lines were cultured with different anti-CD19 antibodies
cross-linked with a 2-fold excess of goat-anti-mouse ADC
(187.1-vcMMAF8). Cultures were incubated for 96 hours and labeled
with 50 .mu.M resazurin. There was no effect of 187.1-vcMMAF on the
growth of any of the cell lines tested. Values are the mean.+-.SD
of four replicates within a single experiment.
Example 4
[0589] Antitumor activity of anti-CD19 antibody-drug conjugate
compounds on Ramos tumor model in SCID mice was determined. The
results generally show that murine and chimeric BU12 antibody drug
conjugates had poor activity as compared to other chimeric
anti-CD19 antibody drug conjugates and as compared to humanized
BU12 antibody drug conjugates. See FIGS. 3, 4, 5, 7 and 8.
Example 5
[0590] Variants of humanized BU12 antibody, in which amino acid
residues in the Fc domain of IgG.sub.1 known to be important for
binding to Fc.gamma.R can be mutated to impair binding to one or
more Fc.gamma.R, can be generated using standard molecular biology
techniques.
[0591] For example, IgG1v1 contains the following mutations:
E233P:L234V:L235A, according to the Kabat numbering scheme. The
amino acid sequence of IgG1V1 is shown in SEQ ID NO:35.
[0592] Further Fc domain variants of humanized anti-CD19 antibodies
can be similarly generated, including, for example, Fc domain
variants with one or more non-conservative amino acid
substitutions, introduction of one or more cysteine residues, or
introduction of one or more sites for N-linked glycosylation, in or
in proximity to the Fc domain involved in the binding interaction
to one or more Fc.gamma. receptors.
Example 6
[0593] Preparation of a hBU12 Antibody Drug Conjugate
[0594] One hundred thirty milligrams of the hBU12 mAb (Lot #'s
PR208 (69 mg) and 1033154 (100 mg)) were combined and concentrated
to provide 141 mg at a concentration of 10.8 mg/mL, based on a
molecular weight of 150 kD and an extinction coefficient of 1.47
AUmLmg.sup.-1cm.sup.-1.
[0595] The auristatins MMAE and MMAF were conjugated to the
purified antibody as follows. The antibody (130 mg, 867 nmol) was
incubated 45 min at 37.degree. C. with 2.17 nmol of TCEP
(representing a 25% excess of reductant for the desired reduction
level of 4 free thiols per antibody) with 1 mM DTPA as a cation
scavenger. The reduction level was determined by performing a
microscale test conjugation with the following test compound:
##STR00034##
[0596] The drug loading distribution was characterized by HIC
chromatography. This mAb exhibited a reduction pattern occasionally
seen with murine antibodies, wherein the distribution is weighted
at 0 and 10 drugs per antibody, with 4- and 6-loaded antibody being
represented at lower levels. The mean drug loading was higher than
desired: 4.9 drugs/Ab. Incremental quantities of DTNB (217 nmol,
then 303.8 nmol) were added to re-oxidize antibody disulfides,
thereby reducing the drug loading level to an assayed level of 4.1
drugs/Ab.
[0597] The partially reduced mAb (97 mg, 647 nmol) was conjugated
with MMAF by addition of 795 .mu.L of DMSO to the approximately 9.0
mL of mAb solution, followed by 203.1 .mu.L of a 19.1 mM DMSO
solution of the following compound, maleimidocaproyl-Val-Cit-MMAF
(3.88 .mu.mol).
##STR00035##
[0598] The conjugation reaction was allowed to proceed for 100
minutes at 0.degree. C. Residual maleimidocaproyl-Val-Cit-MMAF was
quenched by addition of 194 .mu.L of 100 mM N-acetyl cysteine. The
reaction mixture was then dialyzed against 4 L of PBS three times
using a 25000 MWCO membrane at 4.degree. C. to remove DMSO,
unreacted or quenched drug, and other small-molecule contaminants
resulting from the conjugation process, and concentrated. The
product contained 4.1 drugs/Ab
Example 7
[0599] Activity of the Anti-CD19 Auristatin Antibody Drug Conjugate
hBU12-vcMMAE Against Rituximab Sensitive and Resistant Lymphomas
and in CD21 High and Low Lymphomas:
[0600] Materials and Methods
[0601] Flow Cytometric Analysis to Determine CD19 and CD21
Expression Levels on Tumor Cell Lines:
[0602] To evaluate CD19 and CD21 copy numbers on tumor cell lines,
cells were incubated for 30 minutes on ice with PE-conjugated
murine anti-CD19 and anti-CD21 antibodies (BD Pharmingen, San
Diego, Calif.), washed with cold staining medium and evaluated with
a Becton Dickison FACScan flow cytometer. Quantitative
determination of CD19 and CD21 on the cell surface was determined
using a DAKO QiFiKit flow cytometric indirect immunofluorescence
assay and murine antibodies as described by the manufacturer (DAKO
A/S, Glostrup, Denmark).
[0603] Saturation Binding Studies to Determine Binding
Affinity:
[0604] Cells were incubated with 10 .mu.g/ml hBU12 or hBU12-vcMMAE
for 0.5 h at 4 C, and washed. One set of cells was transferred to
37.degree. C. and harvested at selected timepoints. For detection,
a secondary PE-conjugated antibody was used and the amount of
remaining surface-bound antibody determined by flow cytometry.
Alternatively, cells were incubated on ice with
AlexaFluor488-labeled hBU12 antibody or drug conjugates for 1 h,
washed with cold PBS, and binding assessed with a Becton Dickison
FACScan flow cytometer. The apparent Kd values were determined
using the One Site Binding algorithm from Prism (GraphPad Software,
San Diego, Calif.).
[0605] CD19 Internalization Kinetic Studies:
[0606] To generate radiolabeled antibody-drug conjugates, custom
synthesized [3H]-vcMMAE (24.7 Ci/mmol, Moravek Biochemicals, Brea,
Calif.) was used to prepare the radiolabeled hBU12-vcMMAE
conjugate. Calculations of radioactivity were made. The amount of
free drug found inside of the cells from 1 mL of culture was added
to the amount of free drug detected in 1 mL of culture medium and
this value was used to determine the concentration of total drug
released in the cell culture. Triplicate results were averaged and
the standard deviation for those values was calculated using the
STDEVPA function in Microsoft Excel.
[0607] Lysosomal Co-Localization Studies of hBU12 and
hBU12-ADCs:
[0608] Ramos cells were incubated with 1 ug/ml hBU12 or hBU12-ADCs
on ice or for 20 minutes or 4 hours at 37.degree. C. After the
incubation, the cells were washed with cold PBS to remove unbound
antibody or ADC and then fixed and permeabilized with BD
Cytofix/Cytoperm (BD Biosciences, San Jose, Calif.). The antibody
and ADCs were detected with AlexaFluor-488 labeled goat anti-human
IgG (Molecular Probes, Eugene, Oreg.). Lysosomal compartments were
visualized by staining with AlexaFluor647-labeled LAMP-1 antibody
(mouse CD107, BD Biosciences). Nuclear compartments were stained
with DAPI (4', 6-diamidino-2-phenylindole, Roche, Basel,
Switzerland). Fluorescence images were acquired with a Carl Zeiss
Axiovert 200M microscope.
[0609] Cytotoxicity and Growth Arrest Assays:
[0610] Tumor cells were incubated with hBU12 and the drug
conjugates for 96 h. Cell viability was measured by Alamar Blue
(Biosource International, Camarillo, Calif.) dye reduction as
previously reported (Doronina, 2003 #1834). Cells were incubated
for 4 h with the dye and dye reduction measured on a Fusion HT
fluorescent plate reader (Perkin Elmer, Waltham, Mass.). Results
are reported as IC50, the concentration of compound needed to yield
a 50% reduction in viability compared to vehicle-treated cells
(control=100%). For growth arrest and apoptosis studies, cells were
first treated with the antibody and ADCs and then processed using
the Annexin V-FITC Apoptosis Detection kit (BD Pharmingen),
according to the manufacturer's directions. For analysis of cell
cycle position following exposure to ADCs, the cells were labeled
for 20 minutes with bromodeoxyuridine (BrdUrd, Sigma, St. Louis,
Mo.). Nascent DNA synthesis was detected using an anti-BrdUrd
antibody (BD Biosciences) and total DNA content was detected with
propidium iodide (PI). Cells were then analyzed by flow
cytometry.
[0611] In Vivo Model of Subcutaneous Lymphomas and Disseminated
Human Leukemias:
[0612] Localized, subcutaneous and disseminated models of B cell
lymphomas were established in SCID mice. For the subcutaneous
model, 5.times.10.sup.6 lymphoma cells were implanted into the
right flank of female mice. hBU12 and -ADCs or a control compound
were administered when tumor volumes reached 100 mm.sup.3. Tumor
size was monitored at least twice weekly. To establish disseminated
disease, 1.times.10.sup.5 Nalm6 cells or RS4; 11 cells in 0.2 ml
PBS were injected into the lateral tail vein of C.B.-17 SCID mice
(Harlan, Indianapolis, Ind.). Mice were treated with test compounds
7 days after cells injection and monitored at least twice per week.
Mice were terminated when they exhibited signs of disease including
weight loss of 15-20%, hunched posture and lack of grooming,
cranial swelling and hind limb paralysis. Treatment schedules are
as indicated in the figure legends.
[0613] Statistical Analysis:
[0614] Tumor quadrupling or triplication times (as indicated) were
chosen as time to endpoint (TTE), which were determined by using a
non-linear regression analysis for exponential growth of each
individual tumor growth data set from each experimental animal. The
tumor quadrupling time was calculated based on the tumor volume at
the beginning of treatment. Animals that did not reach the endpoint
were assigned a TTE value equal to the last day of the study. % TGD
(tumor growth delay) reflects the delay in reaching TTE relative to
control treated tumors, which was determined by using the formula:
% TGD=[(T-C)/C].times.100, where T and C are the median times in
days for treated and control groups, to reach TTE, using the start
of treatment as day 1. Statistical analysis and graphic
presentations were conducted using Graphpad Prism Software version
4.01 (Graphpad, San Diego, Calif.). Median tumor growth curves show
group median tumor volumes as a function of time. The Log rank test
was used to analyze the significance of the differences between TTE
of treated and control tumor groups, with differences deemed
significant (*) at 0.01<P<0.05, and highly significant (**)
at P<0.01. In a CR response, the tumor volume is less than 13.5
mm.sup.3 for three consecutive measurements during the course of
the study. A durable response (DR) is defined as complete absence
of palpable tumor during the entire experiment. Standard Pearson
correlation analysis (two tailed) was employed, using a 95%
confidence interval, to determine significant correlations between
CD19 and CD21 expression levels and in vitro cytotoxicity.
[0615] Development of Rituxan Resistant Ramos and Raji Tumors:
[0616] Parental cells were implanted into 40 SCID mice at a
concentration of 5.times.10.sup.6 cells per mouse. 2 days following
cell implant, mice were treated with rituximab at 8 mg/kg every
other day for a total of 9 doses. Out of the 40 mice, roughly 6
developed tumors, when the tumors were approximately 300-400 mm3,
the mice were euthanized and the tumors were collected aseptically.
Tumors were made into a single cell suspension through
disassociation through a nylon filter. While in culture the cells
were continuously exposed to various levels of rituximab up to 100
ug/ml. Cell viability was verified several times per week. After
one week in culture the cells were implanted into 30 SCID mice. Two
days after implant the mice were treated with rituximab at 12 mg/kg
in the schedule as before. The in vitro and in vivo selection was
repeated once more in 10 SCID mice. The resulting tumors were
processed into single cell suspension and frozen in liquid
nitrogen. The Raji R2 and Raji H4 cell lines were generated as
described in Czuczamn et al., Clin Cancer Res. 2008;
14:1561-1570.
[0617] Pharmacokinetic Characteristics of hBU12-vcMMAE(4)
Conjugates:
[0618] Single doses of hBU12-vcMMAE were administered
intra-peritoneally to naive SCID mice. The serum samples were
collected at scheduled intervals over a period of 11 weeks to
obtain composite pharmacokinetic profiles. The samples were
analyzed for antibody drug conjugate concentrations by a qualified
multiplex bead-capture assay using an anti-MMAE antibody. The
pharmacokinetic analysis was done using non-compartmental and
compartmental methods.
[0619] Results
[0620] Lack of Correlation Between CD19 and CD21 Expression and
Potency of hBU12-vcMMAE Against ALL, CLL and NHL Tumor Cell Lines
Grown in Culture:
[0621] In order to determine the potency of hBU12-vcMMAE, CD19
positive lymphoma and leukemia cells representing Burkitt's
lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular
lymphomas (FL), and acute lymphocytic leukemia (ALL) were exposed
to increasing concentrations of hBU12-vcMMAE. In addition, the cell
surface copy numbers of CD19 and CD21 were determined in order to
study a potential correlation between expression of these genes
with anti-tumor activity. Potent cytotoxic activity of the
hBU12-vcMMAE conjugate was noticed in 15 out of 17 CD19 positive
tumor cell lines tested. The T-cell lymphoma cell line Jurkat was
used as CD19 negative control cell line. The absence of activity of
hBU12-vcMMAE on these control cells suggested that the anti-tumor
activity was target dependent. A lack of significant correlations
between ADC potency and the levels of CD19 (p=0.45, R2=0.038) and
CD21 (p=0.55, R2=0.028) expression was noticed (data not shown). In
addition, similar potent cytotoxic effects of hBU12-vcMMAE against
ALL cell lines were found. In sum, the data demonstrates that CD19
and CD21 expression levels and the tumor subtypes are insufficient
to predict the sensitivities of lymphoma and leukemia cell lines
towards auristatin based ADCs.
[0622] Internalization Kinetics and Intracellular Trafficking of
hBU12-vcMMAE in NHL Cell Lines:
[0623] A critical parameter previously shown to determine the
anti-tumor effects of certain auristatin based ADCs is the ability
of the target antigen to internalize and to translocate to the
lysosomal compartment following ligation by the antibody. To study
these processes, CD21 low (Ramos and SUDHL-4) and CD21 high tumor
cell lines (Raji, Daudi) were incubated with hBU12-vcMMAE and the
internalization kinetics by fluorescence activated cell sorting
(FACS) was determined. As shown in FIG. 15, hBU12 and hBU12-vcMMAE
conjugates internalized rapidly in CD21 low Ramos cells, and
>50% of the compounds internalized within 60 minutes post
incubation. Somewhat slower internalization kinetics of
hBU12-vcMMAE were found in CD2 1high Raji and Daudi cells. However,
the small differences in internalization kinetics did not
significantly affect potency, and comparable IC50 values between
CD21 low Ramos cells and CD21 high lymphomas were found. Combined,
the findings demonstrate that the internalization kinetics of
hBU12-vcMMAE on different tumor cell lines does not correlate with
cytotoxicity in vitro. Intracellular trafficking of hBU12 and
hBU12-vcMMAE conjugates in NHL cell lines was investigated. For
this purpose, CD21low Ramos and SUDHL4 cells were incubated with
either naked antibody or hBU12-vcMMAE. Co-immunofluorescence
studies revealed that the majority of internalized hBU12 localized
to lysosomes, starting as early as 15 minutes post incubation.
Comparable subcellular translocation of hBU12 and conjugates to the
lysosomal compartment was observed between CD21 low Ramos or HT and
CD21 high Daudi and Raji cells (data not shown). Combined, the
findings demonstrate that internalization kinetics alone are
insufficient to explain the differences in hBU12-vcMMAE potencies
against different NHL cell lines.
[0624] Free Drug Release by hBU12-vcE in Rituximab Sensitive and
Resistant, CD21 High and Low Lymphoma Cell Lines:
[0625] MMAE interferes with microtubule stability in the
cytoplasmic compartment and thus, the amounts of active, free MMAE
drug released in tumor cells is critical for anti-lymphoma effects.
To investigate theses aspects, CD21 high Daudi and CD21 high,
rituximab resistant Raji R2 and Raji 4H cells were incubated with
hBU12-vcMMAE and the levels of free drug released with CD21 low
Ramos cells were compared. The cellular release of free, active
drug was quantified by combining the radioactivity that was
retained within cells and that had escaped into the supernatant
over time. There was no difference in free drug release between
CD21 low cells (Ramos) and CD21 high cells (Daudi, Table 2).
Therefore, it is unlikely that variations in free drug release
account for the >50 fold differences in the IC50 values between
these different lymphoma cell lines. In conclusion, high CD21
levels may only minimally interfere with the intracellular release
of free drug from internalized hBU12-vcMMAE.
[0626] Efficacy of hBU12 conjugates in models of NHL and ALL
hBU12-vcMMAE was tested in single dose and multi dose experiments
using different NHL cell lines xenografted into SCID mice (FIG.
16A-E and data not shown). When tested against Burkitt's lymphoma,
7/8 durable responses were observed at the 3 mg/kg hBU12-vcMMAE
dose, administered q4dx4 (FIG. 16A)). When tested against DOHH2
tumor (Follicular lymphoma), significant inhibition of tumor growth
rates as illustrated by 2/10 DRs at the 3 mg/kg dose level (FIG.
16B) were observed. When tested against SUDHL4 lymphomas (DLBCL),
there was also a significant inhibition of tumor growth rates (FIG.
16C). In the disseminated RS4; 11 model representing ALL, a
significant increase in survival of mice treated with hBU12-vcMMAE
was observed, resulting in a delay of disease onset from .about.45
days in control or untreated animals to >90 days in mice treated
with 3 mg/kg of vcE conjugates (FIG. 16D). Similar observations
were made when testing hBU12-vcMMAE in a second model of
disseminated ALL (Nalm6), where single dose administration resulted
in 30-60% durable responses FIG. 16E). Combined, these data
demonstrate potent anti-tumor effects of hBU12-vcMMAE in different
models of NHL and ALL, irrespective of their CD21 expression
status.
[0627] Antitumor Activity hBU12-vcE Against Rituximab Resistant
Lymphomas:
[0628] In order to develop a preclinical models that mimick the
refractoriness of NHL tumors to rituximab treatment, Ramos tumor
cells were generated that were rendered refractory by repeated in
vitro and in vivo passaging of tumor cells in mice, concurrent with
rituximab treatment as described in materials and methods. To
determine the expression levels of CD20 and CD19, cells from
R-Ramos tumors were isolated and comparable levels of CD20 and CD19
expression in rituximab resistant Ramos tumors were found (FIG.
17C). Compared with high doses of rituximab (12 mg/kg, q4dx4),
hBU12-vcMMAE treatment resulted in a significant difference in %
tumor growth delay (TGD) between the parental and rituximab
resistant tumors. Importantly, similar antitumor activities by
hBU12-vcMMAE in rituximab sensitive and resistant cell lines were
found, suggesting that the mechanism rendering NHL cells resistant
to rituximab does interfere with hBU12-vcMMAE potency. To further
validate these findings, hBU12-vcMMAE was tested in conjunction
with two additional, rituximab resistant NHL cell lines described
previously (Raji 2R and 4RH 27; FIG. 17D and data not shown), and
both cell lines were shown to express similar levels of CD19 and
CD20 compared to the parental clone. In support of the previous
findings testing rituximab resistant Ramos tumors, hBU12-vcMMAE
treatment induced similar durable response compared to the
rituximab sensitive parental Raji clone. In conclusion, potent
anti-tumor activities for CD19-ADCs on rituximab resistant cell
lines were observed.
Example 8
[0629] Role of Effector Cells in Mediating Therapeutic Effects of
the Humanized Anti-CD19 Antibody hBU12 in Preclinical Models of NHL
and ALL:
[0630] The ability of the humanized anti-CD19 antibody hBU12 to
induce CDC, ADCC and ADCP against human lymphoma and leukemia cell
lines was investigated. Potent ADCC and ADCP was found and ADCC was
slightly reduced when hBU12 was conjugated to the vcMMAE drug
linker. When tested on activated, human primary B-cell isolates,
direct anti-proliferative effects of the hBU12 and conjugates was
observed. A lack of CDC anti-tumor effects was noticed for all
hBU12 compounds. To determine the relevance of effector cell
mediates activities for therapeutic activity of hBU12, human
lymphoma and leukemia cells were implanted either subcutaneously
into SCID mice or via tail vein injections (disseminated model).
The most potent anti-tumor effects were observed in disseminated
models, consistent with the notion that the access of effector
cells to the tumor cells is less limiting in the disseminated model
(FIGS. 18 and 19). In order to identify the nature of the cells
mediating anti-tumor effects, effector cell ablation experiments
were conducted in a disseminated model of NHL (Ramos). NK cells,
neutrophils or macrophages were selectively depleted and the
effects on tumor growth inhibition of hBU12 in tumor bearing mice
were measured. Ablation of macrophage and neutrophils almost
completely abolished the anti-tumor effects of hBU12, while
depletion of NK cells was associated with a moderate decrease in
activity. In conclusion, the findings demonstrate that hBU12
induced anti-tumor effects via effector cell mediated ADCP and ADCC
activities.
[0631] Material and Methods
[0632] For in vivo depletion studies, rabbit anti-asialo-GM-1
antibody was obtained from Wako Pure Chemical Industries, Ltd.
(Richmond, Va.), rat anti-mouse-Gr-1 antibody was obtained from BD
Biosciences (San Diego, Calif.). Liposome-encapsulated clodronate
(CEL) was prepared as previously described(Van Rooijen and Sanders
1994). Clodronate was a gift of Roche Diagnostics GmbH (Mannheim,
Germany). Tumor-bearing mice were depleted of effector cells using
specific antibody or CEL as described previously (Van Rooijen and
Sanders 1994; van Rooijen and Sanders 1997; McEarchern, Oflazoglu
et al. 2007). Natural killer (NK) cells were depleted by i.p.
injection of anti-asialo-GM 1 (1.25 mg/kg). Mice were given a total
of 3 doses once every 5 days, beginning the day of tumor cell
implantation. Macrophages were depleted by i.p. injection of CEL
(100.rho.l/10 gr) on the day of tumor injection and every 3 days
thereafter for a total of 5 doses. Cell depletion was confirmed by
flow cytometric analysis of splenocytes, lymph nodes and blood
(data not shown).
TABLE-US-00015 SEQUENCE LISTING SEQ ID NO: 1: Met Gly Arg Leu Thr
Ser Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr Val Leu Ser SEQ ID NO:
2: (CDR regions are underlined. Kabat positions 75, 79, 81, 82,
82A, 82B, 82C, and 89 are in bold font. Residues that determine CDR
structure have an asterix* to their right side) Gln Val Thr Leu Arg
Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys
Thr Phe* Ser Gly* Phe* Ser Leu* Ser Thr Ser Gly Met Gly Val* Gly
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp
Trp Asp Asp* Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Leu Thr
Ile Ser Lys* Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr Asn
Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg* Met Glu Leu
Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser SEQ ID NO: 3: Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys SEQ ID NO: 4: (CDR
regions are underlined. Kabat positions 75, 79, 81, 82, 82A, 82B,
82C, and 89 are in bold font) Gln Val Thr Leu Arg Glu Ser Gly Pro
Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly
Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro
Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg
Tyr Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser
Asn Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr
Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 5: (CDR regions are
underlined. Kabat positions 75, 79, 81, 82, 82A, 82B, 82C, and 89
are in bold font) Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val
Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu
Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala
Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val
Phe Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys
Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser SEQ ID NO: 6: (CDR regions are underlined.
Kabat positions 75, 79, 81, 82, 82A, 82B, 82C, and 89 are in bold
font) Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr
Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp
Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Lys
Ile Ala Ser Val Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Met
Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser SEQ ID NO: 7: (CDR regions are underlined. Kabat
positions 75, 79, 81, 82, 82A, 82B, 82C, and 89 are in bold font)
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr
Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met
Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala
His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg
Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr Met Thr
Asn Met Asp Pro Val Asp Thr Ala Ala Tyr Tyr Cys Ala Arg Met Glu Leu
Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser SEQ ID NO: 8: Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
Gln Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu
Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly
Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val
Phe Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Ala Tyr Tyr Cys
Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser SEQ ID NO: 9: (CDR regions are underlined.
Kabat positions 24, 27, 29, 71, 75, 78, 79, and 89 are in bold
font. Residues that determine CDR structure have an asterix* to
their right side) Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val* Ser Gly* Gly* Ser
Ile* Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly
Lys Gly Leu Glu Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala Leu Lys Ser Arg Val Thr Ile Ser Val* Asp Thr Ser Lys
Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr Cys Ala Arg* Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 10: (CDR regions
are underlined. Kabat positions 24, 27, 29, 71, 75, 78, 79, and 89
are in bold font) Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile
Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly
Leu Glu Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ala Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser SEQ ID NO: 11: (CDR regions are underlined.
Kabat positions 24, 27, 29, 71, 75, 78, 79, and 89 are in bold
font) Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser
Gln Thr Leu Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp
Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Met
Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser SEQ ID NO: 12: (CDR regions are underlined. Kabat
positions 24, 27, 29, 71, 75, 78, 79, and 89 are in bold font) Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu
Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly
Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly His
Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Val
Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
SEQ ID NO: 13: (CDR regions are underlined. Kabat positions 24, 27,
29, 71, 75, 78, 79, and 89 are in bold font) Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Thr Ser Gly Met Gly Val Gly Trp Ile
Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly His Ile Trp Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Val Thr Ile Ser Val
Asp Thr Ser Ser Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 14:
(CDR regions are underlined. Kabat positions 24, 27, 29, 71, 75,
78, 79, and 89 are in bold font) Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Ile Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln His
Pro Gly Lys Gly Leu Glu Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ala Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Val Phe Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser SEQ ID NO: 15: (CDR regions
are underlined. Kabat positions 24, 27, 29, 71, 75, 78, 79, and 89
are in bold font) Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile
Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly
Leu Glu Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro
Ala Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr Cys
Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser SEQ ID NO: 16 Met Asp Phe Gln Val Gln Ile
Phe Ser Phe Leu Leu Ile Ser Ala Ser Val Ile Met Ser Arg Gly SEQ ID
NO: 17 (CDR regions are underlined. Kabat positions 2, 40, 41, 42,
69, 70, 71,
72, and 83 are in bold font. Residues that determine CDR structure
have an asterix* to their right side) Glu Ile* Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe* Thr Leu Thr Ile
Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser
Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ
ID NO: 18 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys SEQ ID NO: 19 (CDR regions are
underlined. Kabat positions 2, 40, 41, 42, 69, 70, 71, 72, and 83
are in bold font) Glu Asn Val Leu Thr Gln Ser Pro Ala Thr Leu Ser
Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val
Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu
Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ ID NO: 20 (CDR regions
are underlined. Kabat positions 2, 40, 41, 42, 69, 70, 71, 72, and
83 are in bold font) Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser
Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp His Thr Leu Thr Ile Ser Ser Leu Glu Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ ID NO: 21 (CDR
regions are underlined. Kabat positions 2, 40, 41, 42, 69, 70, 71,
72, and 83 are in bold font) Glu Asn Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser
Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp His Thr Leu Thr Ile Ser Ser Leu
Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro
Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ ID NO: 22
(CDR regions are underlined. Kabat positions 2, 40, 41, 42, 69, 70,
71, 72, and 83 are in bold font) Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala
Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ala
Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr
Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ ID NO:
23 (CDR regions are underlined. Kabat positions 2, 40, 41, 42, 69,
70, 71, 72, and 83 are in bold font) Glu Ile Val Leu Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro
Ala Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser His Phe Leu Thr Ile Ser
Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val
Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ ID
NO: 24 (CDR regions are underlined. Kabat positions 2, 40, 41, 42,
69, 70, 71, 72, and 83 are in bold font) Glu Ile Val Leu Thr Gln
Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile
Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Glu Pro Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly Ser
Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg SEQ
ID NO: 25 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser
Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met His Trp Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser Gly
Ser Gly Asn Ser His Phe Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Val
Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Ser
Gly Thr Lys Leu Glu Ile Lys Arg SEQ ID NO: 26 (CDR regions are
underlined. Kabat positions 2, and 71 are in bold font. Residues
that determine CDR structure have an asterix* to their right side)
Glu Ile* Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His
Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Asp Thr
Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe* Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg SEQ ID NO: 27 (CDR regions are
underlined. Kabat positions 2, and 71 are in bold font) Glu Asn Val
Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys Glu Lys Val Thr
Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln
Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Asp Thr Ser Lys Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp His Thr
Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Phe
Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys Arg SEQ ID NO: 28 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu
Val Lys Pro Thr Gln Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn
Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Xa Asn Gln
Val Xb Leu Xc Xd Xe Xf Xg Asp Pro Val Asp Thr Ala Xh Tyr Tyr Cys
Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser wherein Xa is Ser or Lys, Xb is Phe or Val,
Xc is Lys or Thr, Xd is Ile or Met, Xe is Ala or Thr Xf is Ser or
Asn, Xg is Val or Met, and Xh is Ala or Thr. SEQ ID NO: 29 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser
Leu Thr Cys Thr Xa Ser Gly Xb Ser Xc Ser Thr Ser Gly Met Gly Val
Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly His Ile
Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Val Thr
Ile Ser Xd Asp Thr Ser Xe Asn Gln Xf Xg Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Xh Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser wherein
Xa is Phe or Val, Xb is Phe or Gly, Xc is Leu or Ile, Xd is Lys or
Val, Xe is Ser or Lys, Xf is Val or Phe, Xg is Phe or Ser, and Xh
is ala or Val. SEQ ID NO: 30 Glu Xa Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Xb Xc Xd Ala Pro Arg
Leu Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe
Ser Gly Ser Gly Ser Gly Xe Xf Xg Xh Leu Thr Ile Ser Ser Leu Glu Pro
Glu Asp Xi Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg wherein Xa is Asn or
Ile, Xb is Ser or Pro, Xc is Ser or Gly, Xd is Thr or Gln, Xe is
Asn or Thr, Xf is Ser or Asp, Xg is His or Phe, Xh is Phe or Thr,
and Xi is Val or Phe. SEQ ID NO: 31 Glu Xa Val Leu Thr Gln Ser Pro
Asp Phe Gln Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Ser Ala
Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Asp Gln Ser
Pro Lys Leu Leu Ile Lys Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Xb Thr Leu Thr Ile Asn Ser
Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr
Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg wherein Xa
is Asn or Ile and Xb is His or Phe. SEQ ID NO: 32 Gln Val Thr Leu
Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly Trp
Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala His Ile Trp Trp
Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Leu Thr Ile Ser
Lys Asp Thr Ser Xa Asn Gln Val Xb Leu Xc Xd Xe Xf Xg Asp Pro Val
Asp Thr Ala Xh Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe
Asp Tyr Trp Gly Xi Gly Thr Xj Val Thr Val Ser Ser wherein Xa is Ser
or Lys, Xb is Phe or Val, Xc is Lys or Thr, Xd is Ile or Met, Xe is
Ala or Thr Xf is Ser or Asn, Xg is Val or Met, Xh is Ala or Thr, Xi
is Gln or Arg, and Xj is Leu, Thr, or Met. SEQ ID NO: 33 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser
Leu Thr Cys Thr Xa Ser Gly Xb Ser Xc Ser Thr Ser Gly Met Gly Val
Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile Gly His Ile
Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys Ser Arg Val Thr
Ile Ser Xd Asp Thr Ser Xe Asn Gln Xf Xg Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Xh Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser Tyr
Tyr Phe Asp Tyr Trp Gly Xi Gly Thr Xj Val Thr Val Ser Ser wherein
Xa is Phe or Val, Xb is Phe or Gly, Xc is Leu or Ile, Xd is Lys or
Val, Xe is Ser or Lys, Xf is Val or Phe, Xg is Phe or Ser, Xh is
Ala or Val, Xi is Gln or Arg, and Xj is Leu, Thr, or Met. SEQ ID
NO: 34 Glu Xa Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
His Trp Tyr Gln Gln Lys Xb Xc Xd Ala Pro Arg Leu Leu Ile Tyr Asp
Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser
Gly Xe Xf Xg Xh Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Xi Ala Val
Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Xj Gly Thr
Xk Xl Xm Ile Lys Arg wherein Xa is Asn or Ile, Xb is Ser or Pro, Xc
is Ser or Gly, Xd is Thr or
Gln, Xe is Asn or Thr, Xf is Ser or Asp, Xg is His or Phe, Xh is
Phe or Thr, Xi is Val or Phe, Xj is Gln, Pro or Gly, Xk is Lys or
Arg, XI is Leu or Val, and Xm is Glu or Asp. SEQ ID NO: 35 Glu Xa
Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys Glu Lys Val
Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln
Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys Asp Thr Ser Lys Leu
Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Xb
Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys
Phe Gln Gly Ser Val Tyr Pro Phe Thr Phe Gly Xc Gly Thr Xd Xe Xf Ile
Lys Arg wherein Xa is Asn or Ile, Xb is His or Phe, Xc is Gln, Pro
or Gly, Xd is Lys or Arg, Xe is Leu or Val, and Xf is Glu or Asp.
SEQ ID NO: 36
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAP
IEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 37
QMQGVNCTVSSELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCP
RCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVQVHNAKTKPREQQFN
STFRVVSVLTVLHQNWLDGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFT
QKSLSLSPGK SEQ ID NO: 38
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT
VPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT
CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPS
SIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO: 39
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSS-
SLG
TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPPVAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH-
EDP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE-
PQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV-
FSC SVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 40
atgggcaggcttacttcttcattcttgttgctgattgtccctgcatatgtcctgtcc SEQ ID
NO: 41
caggttcagctgcaagagtctggccctgggttggttaagccctcccagaccctcagtctgacttgtactgtgtc-
tgg
gggttcaatcagcacttctggtatgggtgtaggctggattaggcagcacccagggaagggtctggagtggattg-
gac
acatttggtgggatgatgacaagagatataacccagccctgaagagcagagtgacaatctctgtggatacctcc-
aag
aaccagtttagcctcaagctgtccagtgtgacagctgcagatactgctgtctactactgtgctagaatggaact-
ttg gtcctactattttgactactggggccaaggcacccttgtcacagtctcctca SEQ ID NO:
42
gctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccct-
ggg
ctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgc-
aca
ccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttg-
ggc
acccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatc-
ttg
tgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccc-
caa
aacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagac-
cct
gaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagta-
caa
cagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgca-
agg
tctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacag-
gtg
tacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttcta-
tcc
cagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgg-
act
ccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctca-
tgc
tccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga
SEQ ID NO: 43
atggattttcaagtgcagattttcagcttcctgctaatcagtgcctcagtcataatgtccagaggagaaa
SEQ ID NO: 44
ttgttctcacccagtctccagcaaccctgtctctctctccaggggaaagggctaccctgagctgcagtgccagc-
tca
agtgtaagttacatgcactggtaccagcagaagccagggcaggctcccagactcctgatttatgacacatccaa-
act
ggcttctggtattccagcaaggttcagtggcagtgggtctggaacagattttacactcacaatcagcagcctgg-
agc
cagaggatgttgctgtctattactgttttcaggggagtgtatacccattcacttttggccaagggacaaagttg-
gaa atcaaaaga SEQ ID NO: 45
actgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgt-
gtg
cctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaact-
ccc
aggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagca-
gac
tacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaa-
cag gggagagtgttag SEQ ID NO: 46 Thr Ser Gly Met Gly Val Gly SEQ ID
NO: 47 His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala Leu Lys
Ser SEQ ID NO: 48 Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr SEQ ID
NO: 49 Ser Ala Ser Ser Ser Val Ser Tyr Met His SEQ ID NO: 50 Asp
Thr Ser Lys Leu Ala Ser SEQ ID NO: 51 Phe Gln Gly Ser Val Tyr Pro
Phe Thr
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 61 <210> SEQ ID NO 1 <211> LENGTH: 19 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 leader sequence -
heavy chain region <400> SEQUENCE: 1 Met Gly Arg Leu Thr Ser
Ser Phe Leu Leu Leu Ile Val Pro Ala Tyr 1 5 10 15 Val Leu Ser
<210> SEQ ID NO 2 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable
Region (VH2-70/JH4 germline) - Variant HA <400> SEQUENCE: 2
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5
10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys
Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met
Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Met Glu
Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 3 <211>
LENGTH: 330 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Constant domain (IgG1) <400> SEQUENCE: 3 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150
155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275
280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330 <210> SEQ ID NO 4 <211> LENGTH: 120 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable
Region (VH2-70/JH4 germline) - Variant HB <400> SEQUENCE: 4
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5
10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys
Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met
Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Met Glu
Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 5 <211>
LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH2-70/JH4 germline) - Variant HC
<400> SEQUENCE: 5 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu
Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser
Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Phe
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90
95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ
ID NO 6 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH2-70/JH4 germline) - Variant HD <400> SEQUENCE: 6 Gln Val
Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Ile Ala Ser Val Asp Pro
Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 7 <211> LENGTH: 120
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain
Variable Region (VH2-70/JH4 germline) - Variant HE <400>
SEQUENCE: 7 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro
Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro
Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met
Thr Asn Met Asp Pro Val Asp Thr Ala Ala Tyr Tyr 85 90 95 Cys Ala
Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110
Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 8
<211> LENGTH: 120 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Heavy Chain Variable Region (Murine) <400>
SEQUENCE: 8 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro
Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser
Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro
Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp
Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr
Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe Leu Lys Ile
Ala Ser Val Asp Thr Ala Asp Thr Ala Ala Tyr Tyr 85 90 95 Cys Ala
Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110
Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> SEQ ID NO 9
<211> LENGTH: 120 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Heavy Chain Variable Region (VH4-31/JH4 germline) -
Variant HF <400> SEQUENCE: 9 Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Gly Ser Ile Ser Thr Ser 20 25 30 Gly Met Gly Val
Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile
Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60
Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65
70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 10 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable
Region (VH4-31/JH4 germline) - Variant HG <400> SEQUENCE: 10
Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5
10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr
Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys
Gly Leu Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Lys
Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu
Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 11 <211>
LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH4-31/JH4 germline) - Variant HH
<400> SEQUENCE: 11 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Phe
Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His
Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85
90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 12 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH4-31/JH4 germline) - Variant HI <400> SEQUENCE: 12 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Lys Asp Thr
Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 13 <211> LENGTH:
120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH4-31/JH4 germline) - Variant HJ
<400> SEQUENCE: 13 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His
Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Ser Asn Gln Phe 65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85
90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 14 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH4-31/JH4 germline) - Variant HK <400> SEQUENCE: 14 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Phe Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 15 <211> LENGTH:
120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH4-31/JH4 germline) - Variant HL
<400> SEQUENCE: 15 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His
Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr 85
90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 16 <211> LENGTH: 22 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Light Chain Region - Leader
Sequence <400> SEQUENCE: 16 Met Asp Phe Gln Val Gln Ile Phe
Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly
20 <210> SEQ ID NO 17 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Variable
Region (VL-L6/Jk2 germline) - Variant LA <400> SEQUENCE: 17
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID NO 18
<211> LENGTH: 106 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain Constant domain (Kappa domain)
<400> SEQUENCE: 18 Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85
90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210>
SEQ ID NO 19 <211> LENGTH: 107 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Light Chain Variable Region
(VL-L6/Jk2 germline) - Variant LB <400> SEQUENCE: 19 Glu Asn
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe Gln
Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 <210> SEQ ID NO 20 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain Variable Region (VL-L6/Jk2 germline) - Variant LC
<400> SEQUENCE: 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys
Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser
Gly Thr Asp His Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85
90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 21 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Variable
Region (VL-L6/Jk2 germline) - Variant LD <400> SEQUENCE: 21
Glu Asn Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp His Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID NO 22
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain Variable Region (VL-L6/Jk2 germline) -
Variant LE <400> SEQUENCE: 22 Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Ser Ser Thr Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr
Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65
70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro
Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
105 <210> SEQ ID NO 23 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Variable
Region (VL-L6/Jk2 germline) - Variant LF <400> SEQUENCE: 23
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser His Phe Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID NO 24
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain Variable Region (VL-L6/Jk2 germline) -
Variant LG <400> SEQUENCE: 24 Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr
Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65
70 75 80 Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro
Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100
105 <210> SEQ ID NO 25 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Variable
Region (Murine) <400> SEQUENCE: 25 Glu Asn Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp
Tyr Gln Gln Lys Ser Ser Thr Ser Pro Lys Leu Trp Ile Tyr 35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Phe Ser Gly Ser 50
55 60 Gly Ser Gly Asn Ser His Phe Leu Thr Ile Ser Ser Met Glu Ala
Glu 65 70 75 80 Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr
Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
100 105 <210> SEQ ID NO 26 <211> LENGTH: 107
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic hBU12 Light Chain
Variable Region (VL-A10/Jk2 germline) - Variant LH <400>
SEQUENCE: 26 Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val
Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln
Ser Pro Lys Leu Leu Ile Lys 35 40 45 Asp Thr Ser Lys Leu Ala Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe
Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID
NO 27 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Light Chain Variable Region
(VL-A10/Jk2 germline) - Variant LI <400> SEQUENCE: 27 Glu Asn
Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
Lys 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp His Thr Leu Thr Ile Asn
Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Phe Gln
Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 <210> SEQ ID NO 28 <211>
LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Consensus sequence for Heavy Chain Variable Region (VH2-70/JH4
germline) <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (77)..(77) <223> OTHER INFORMATION: Xaa
is Ser or Lys <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa
is Phe or Val <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (83)..(83) <223> OTHER INFORMATION: Xaa
is Lys or Thr <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (84)..(84) <223> OTHER INFORMATION: Xaa
is Ile or Met <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (85)..(85) <223> OTHER INFORMATION: Xaa
is Ala or Thr <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (86)..(86) <223> OTHER INFORMATION: Xaa
is Ser or Asn <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (87)..(87) <223> OTHER INFORMATION: Xaa
is Val or Met <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa
is Ala or Thr <400> SEQUENCE: 28 Gln Val Thr Leu Arg Glu Ser
Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp
Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55
60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Xaa Asn Gln Val
65 70 75 80 Xaa Leu Xaa Xaa Xaa Xaa Xaa Asp Pro Val Asp Thr Ala Xaa
Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 29 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Heavy Chain Variable Region (VH4-31/JH4 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa is Phe or Val
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa is Phe or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (29)..(29) <223> OTHER INFORMATION: Xaa is Leu or
Ile <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (73)..(73) <223> OTHER INFORMATION: Xaa is Lys or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (77)..(77) <223> OTHER INFORMATION: Xaa is Ser or
Lys <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(80) <223> OTHER INFORMATION: Xaa is Val or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa is Phe or
Ser <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa is Ala or
Val <400> SEQUENCE: 29 Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Xaa Ser Gly Xaa Ser Xaa Ser Thr Ser 20 25 30 Gly Met Gly Val Gly
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly
His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu
Lys Ser Arg Val Thr Ile Ser Xaa Asp Thr Ser Xaa Asn Gln Xaa 65 70
75 80 Xaa Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa Tyr
Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 30 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Light Chain Variable Region (VL-L6/Jk2 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is Asn or Ile
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (39)..(39) <223> OTHER INFORMATION: Xaa is Ser or
Pro <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (40)..(40) <223> OTHER INFORMATION: Xaa is Ser or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (41)..(41) <223> OTHER INFORMATION: Xaa is Thr or
Gln <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (68)..(68) <223> OTHER INFORMATION: Xaa is Asn or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (69)..(69) <223> OTHER INFORMATION: Xaa is Ser or
Asp <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa is His or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (71)..(71) <223> OTHER INFORMATION: Xaa is Phe or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (82)..(82) <223> OTHER INFORMATION: Xaa is Val or
Phe <400> SEQUENCE: 30 Glu Xaa Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln
Lys Xaa Xaa Xaa Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser
Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Xaa Xaa Xaa Xaa Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70
75 80 Asp Xaa Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe
Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 31 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Light Chain Variable Region (VL-A10/Jk2 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is Asn or Ile
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa is His or
Phe <400> SEQUENCE: 31 Glu Xaa Val Leu Thr Gln Ser Pro Asp
Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln
Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys 35 40 45 Asp Thr Ser
Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Asp Xaa Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe
Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 32 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Heavy Chain Variable Region (VH2-70/JH1-6 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(77)..(77) <223> OTHER INFORMATION: Xaa is Ser or Lys
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa is Phe or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (83)..(83) <223> OTHER INFORMATION: Xaa is Lys or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (84)..(84) <223> OTHER INFORMATION: Xaa is Ile or
Met <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (85)..(85) <223> OTHER INFORMATION: Xaa is Ala or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (86)..(86) <223> OTHER INFORMATION: Xaa is Ser or
Asn <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (87)..(87) <223> OTHER INFORMATION: Xaa is Val or
Met <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa is Ala or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (112)..(112) <223> OTHER INFORMATION: Xaa is Gln or
Arg <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (115)..(115) <223> OTHER INFORMATION: Xaa is Leu,
Thr, or Met <400> SEQUENCE: 32 Gln Val Thr Leu Arg Glu Ser
Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp
Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55
60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Xaa Asn Gln Val
65 70 75 80 Xaa Leu Xaa Xaa Xaa Xaa Xaa Asp Pro Val Asp Thr Ala Xaa
Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp
Tyr Trp Gly Xaa 100 105 110 Gly Thr Xaa Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 33 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Heavy Chain Variable Region (VH4-31/JH1-6 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa is Phe or Val
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa is Phe or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (29)..(29) <223> OTHER INFORMATION: Xaa is Leu or
Ile <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (73)..(73) <223> OTHER INFORMATION: Xaa is Lys or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (77)..(77) <223> OTHER INFORMATION: Xaa is Ser or
Lys <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(80) <223> OTHER INFORMATION: Xaa is Val or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa is Phe or
Ser <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa is Ala or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (112)..(112) <223> OTHER INFORMATION: Xaa is Gln or
Arg <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (115)..(115) <223> OTHER INFORMATION: Xaa is Leu,
Thr, or Met <400> SEQUENCE: 33 Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Xaa Ser Gly Xaa Ser Xaa Ser Thr Ser 20 25 30 Gly Met Gly
Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp
Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55
60 Leu Lys Ser Arg Val Thr Ile Ser Xaa Asp Thr Ser Xaa Asn Gln Xaa
65 70 75 80 Xaa Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa
Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp
Tyr Trp Gly Xaa 100 105 110 Gly Thr Xaa Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 34 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Light Chain Variable Region (VL-L6/Jk1-5 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is Asn or Ile
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (39)..(39) <223> OTHER INFORMATION: Xaa is Ser or
Pro <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (40)..(40) <223> OTHER INFORMATION: Xaa is Ser or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (41)..(41) <223> OTHER INFORMATION: Xaa is Thr or
Gln <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (68)..(68) <223> OTHER INFORMATION: Xaa is Asn or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (69)..(69) <223> OTHER INFORMATION: Xaa is Ser or
Asp <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa is His or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (71)..(71) <223> OTHER INFORMATION: Xaa is Phe or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (82)..(82) <223> OTHER INFORMATION: Xaa is Val or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (99)..(99) <223> OTHER INFORMATION: Xaa is Gln, Pro
or Gly <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (102)..(102) <223> OTHER INFORMATION:
Xaa is Lys or Arg <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (103)..(103) <223> OTHER
INFORMATION: Xaa is Leu or Val <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (104)..(104) <223>
OTHER INFORMATION: Xaa is Glu or Asp <400> SEQUENCE: 34 Glu
Xaa Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30 His Trp Tyr Gln Gln Lys Xaa Xaa Xaa Ala Pro Arg Leu Leu
Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Xaa Xaa Xaa Xaa Leu Thr Ile
Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Xaa Ala Val Tyr Tyr Cys Phe
Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Xaa Gly Thr Xaa
Xaa Xaa Ile Lys Arg 100 105 <210> SEQ ID NO 35 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Consensus sequence for Light Chain Variable Region (VL-A10/Jk1-5
germline) <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa
is Asn or Ile <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa
is His or Phe <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (99)..(99) <223> OTHER INFORMATION: Xaa
is Gln, Pro or Gly <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (102)..(102) <223> OTHER
INFORMATION: Xaa is Lys or Arg <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (103)..(103) <223>
OTHER INFORMATION: Xaa is Leu or Val <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (104)..(104)
<223> OTHER INFORMATION: Xaa is Glu or Asp <400>
SEQUENCE: 35 Glu Xaa Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val
Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln
Ser Pro Lys Leu Leu Ile Lys 35 40 45 Asp Thr Ser Lys Leu Ala Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp
Xaa Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe
Gly Xaa Gly Thr Xaa Xaa Xaa Ile Lys Arg 100 105 <210> SEQ ID
NO 36 <211> LENGTH: 326 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Constant Domain (IgG2)
<400> SEQUENCE: 36 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80
Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala
Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg
Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210
215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser
Leu Ser Pro Gly Lys 325 <210> SEQ ID NO 37 <211>
LENGTH: 290 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Constant Domain (IgG3) <400> SEQUENCE: 37 Gln Met
Gln Gly Val Asn Cys Thr Val Ser Ser Glu Leu Lys Thr Pro 1 5 10 15
Leu Gly Asp Thr Thr His Thr Cys Pro Arg Cys Pro Glu Pro Lys Ser 20
25 30 Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser
Cys 35 40 45 Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro Glu Pro Lys
Ser Cys Asp 50 55 60 Thr Pro Pro Pro Cys Pro Arg Cys Pro Ala Pro
Glu Leu Leu Gly Gly 65 70 75 80 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 85 90 95 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 100 105 110 Asp Pro Glu Val Gln
Phe Lys Trp Tyr Val Asp Gly Val Gln Val His 115 120 125 Asn Ala Lys
Thr Lys Pro Arg Glu Gln Gln Phe Asn Ser Thr Phe Arg 130 135 140 Val
Val Ser Val Leu Thr Val Leu His Gln Asn Trp Leu Asp Gly Lys 145 150
155 160 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 165 170 175 Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 180 185 190 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 195 200 205 Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 210 215 220 Glu Ser Ser Gly Gln Pro Glu
Asn Asn Tyr Asn Thr Thr Pro Pro Met 225 230 235 240 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 245 250 255 Lys Ser
Arg Trp Gln Gln Gly Asn Ile Phe Ser Cys Ser Val Met His 260 265 270
Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu Ser Leu Ser Pro 275
280 285 Gly Lys 290 <210> SEQ ID NO 38 <211> LENGTH:
327 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Constant Domain (IgG4) <400> SEQUENCE: 38 Ala Ser
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15
Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20
25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser
Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly
Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150
155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275
280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325
<210> SEQ ID NO 39 <211> LENGTH: 330 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain Constant
Domain Variant (IgG1V1) <400> SEQUENCE: 39 Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Pro Val Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165
170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290
295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 40 <211> LENGTH: 57 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain Region -
Leader Sequence <400> SEQUENCE: 40 atgggcaggc ttacttcttc
attcttgttg ctgattgtcc ctgcatatgt cctgtcc 57 <210> SEQ ID NO
41 <211> LENGTH: 360 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH4-31/JH4 germline) - Variant HF <400> SEQUENCE: 41
caggttcagc tgcaagagtc tggccctggg ttggttaagc cctcccagac cctcagtctg
60 acttgtactg tgtctggggg ttcaatcagc acttctggta tgggtgtagg
ctggattagg 120 cagcacccag ggaagggtct ggagtggatt ggacacattt
ggtgggatga tgacaagaga 180 tataacccag ccctgaagag cagagtgaca
atctctgtgg atacctccaa gaaccagttt 240 agcctcaagc tgtccagtgt
gacagctgca gatactgctg tctactactg tgctagaatg 300 gaactttggt
cctactattt tgactactgg ggccaaggca cccttgtcac agtctcctca 360
<210> SEQ ID NO 42 <211> LENGTH: 993 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain Constant
domain (IgG1) <400> SEQUENCE: 42 gctagcacca agggcccatc
ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg
ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa tga 993 <210>
SEQ ID NO 43 <211> LENGTH: 66 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Light Chain Region - Leader
Sequence <400> SEQUENCE: 43 atggattttc aagtgcagat tttcagcttc
ctgctaatca gtgcctcagt cataatgtcc 60 agagga 66 <210> SEQ ID NO
44 <211> LENGTH: 321 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Light Chain Variable Region (VL-L6/Jk2
germline) - Variant LG <400> SEQUENCE: 44 gaaattgttc
tcacccagtc tccagcaacc ctgtctctct ctccagggga aagggctacc 60
ctgagctgca gtgccagctc aagtgtaagt tacatgcact ggtaccagca gaagccaggg
120 caggctccca gactcctgat ttatgacaca tccaaactgg cttctggtat
tccagcaagg 180 ttcagtggca gtgggtctgg aacagatttt acactcacaa
tcagcagcct ggagccagag 240 gatgttgctg tctattactg ttttcagggg
agtgtatacc cattcacttt tggccaaggg 300 acaaagttgg aaatcaaaag a 321
<210> SEQ ID NO 45 <211> LENGTH: 321 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Constant
domain (Kappa domain) <400> SEQUENCE: 45 actgtggctg
caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 60
actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg
120 aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga
gcaggacagc 180 aaggacagca cctacagcct cagcagcacc ctgacgctga
gcaaagcaga ctacgagaaa 240 cacaaagtct acgcctgcga agtcacccat
cagggcctga gctcgcccgt cacaaagagc 300 ttcaacaggg gagagtgtta g 321
<210> SEQ ID NO 46 <211> LENGTH: 7 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain CDR1
<400> SEQUENCE: 46 Thr Ser Gly Met Gly Val Gly 1 5
<210> SEQ ID NO 47 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain CDR2
<400> SEQUENCE: 47 His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> SEQ ID NO 48
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Heavy Chain CDR3 <400> SEQUENCE: 48 Met Glu
Leu Trp Ser Tyr Tyr Phe Asp Tyr 1 5 10 <210> SEQ ID NO 49
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain CDR1 <400> SEQUENCE: 49 Ser Ala
Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> SEQ ID NO 50
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain CDR2 <400> SEQUENCE: 50 Asp Thr
Ser Lys Leu Ala Ser 1 5 <210> SEQ ID NO 51 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain CDR3 <400> SEQUENCE: 51 Phe Gln Gly Ser Val Tyr
Pro Phe Thr 1 5 <210> SEQ ID NO 52 <211> LENGTH: 4
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic linker
<400> SEQUENCE: 52 Gly Phe Leu Gly 1 <210> SEQ ID NO 53
<211> LENGTH: 1410 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic alternative sequence for hBU12 Heavy Chain
<400> SEQUENCE: 53 atgggatgga gctggatctt tcttttcctc
ctgtcaggaa ctgcaggtgt ccattgtcag 60 gttcagctgc aagagtctgg
ccctgggttg gttaagccct cccagaccct cagtctgact 120 tgtactgtgt
ctgggggttc aatcagcact tctggtatgg gtgtaggctg gattaggcag 180
cacccaggga agggtctgga gtggattgga cacatttggt gggatgatga caagagatat
240 aacccagccc tgaagagcag agtgacaatc tctgtggata cctccaagaa
ccagtttagc 300 ctcaagctgt ccagtgtgac agctgcagat actgctgtct
actactgtgc tagaatggaa 360 ctttggtcct actattttga ctactggggc
caaggcaccc ttgtcacagt ctcctcagct 420 agcaccaagg gcccatctgt
cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagctgccc
tgggctgcct ggtcaaggac tacttccctg aacctgtgac agtgtcctgg 540
aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga
600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac
ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg
acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg
tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc
aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg
tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc
960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa
tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca
tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg
tacaccctgc ccccatcccg ggatgagctg 1140 accaagaacc aggtcagcct
gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg
agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260
gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag
1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca
ctacacacag 1380 aagagcctct ccctgtctcc gggtaaatga 1410 <210>
SEQ ID NO 54 <211> LENGTH: 57 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic alternative leader sequence for hBU12
Heavy Chain <400> SEQUENCE: 54 atgggatgga gctggatctt
tcttttcctc ctgtcaggaa ctgcaggtgt ccattgt 57 <210> SEQ ID NO
55 <211> LENGTH: 993 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic alternative sequence for hBU12 Heavy Chain -
Constant domain <400> SEQUENCE: 55 gctagcacca agggcccatc
tgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagctg
ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca
180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg
cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg
tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca
ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc
cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat
gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg
900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa
ccactacaca 960 cagaagagcc tctccctgtc tccgggtaaa tga 993 <210>
SEQ ID NO 56 <211> LENGTH: 469 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic alternative sequence for hBU12 Heavy
Chain <400> SEQUENCE: 56 Met Gly Trp Ser Trp Ile Phe Leu Phe
Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Cys Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Gln Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Thr Ser
Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys 50 55 60 Gly
Leu Glu Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr 65 70
75 80 Asn Pro Ala Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys 85 90 95 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Met Glu Leu Trp Ser
Tyr Tyr Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195
200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315
320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440
445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
450 455 460 Leu Ser Pro Gly Lys 465 <210> SEQ ID NO 57
<211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic alternative leader sequence for hBU12 Heavy Chain
<400> SEQUENCE: 57 Met Gly Trp Ser Trp Ile Phe Leu Phe Leu
Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Cys <210> SEQ ID NO
58 <211> LENGTH: 699 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic alternative sequence for hBU12 Light Chain
<400> SEQUENCE: 58 atgaagttgc ctgttaggct gttggtgctg
atgttctgga ttcctgcttc cagcagtgaa 60 attgttctca cccagtctcc
agcaaccctg tctctctctc caggggaaag ggctaccctg 120 agctgcagtg
ccagctcaag tgtaagttac atgcactggt accagcagaa gccagggcag 180
gctcccagac tcctgattta tgacacatcc aaactggctt ctggtattcc agcaaggttc
240 agtggcagtg ggtctggaac agattttaca ctcacaatca gcagcctgga
gccagaggat 300 gttgctgtct attactgttt tcaggggagt gtatacccat
tcacttttgg ccaagggaca 360 aagttggaaa tcaaaagaac tgtggctgca
ccatctgtct tcatcttccc gccatctgat 420 gagcagttga aatctggaac
tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga 480 gaggccaaag
tacagtggaa ggtggataac gccctccaat cgggtaactc ccaggagagt 540
gtcacagagc aggacagcaa ggacagcacc tacagcctca gcagcaccct gacgctgagc
600 aaagcagact acgagaaaca caaagtctac gcctgcgaag tcacccatca
gggcctgagc 660 tcgcccgtca caaagagctt caacagggga gagtgttag 699
<210> SEQ ID NO 59 <211> LENGTH: 57 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic alternative leader
sequence for hBU12 Light Chain <400> SEQUENCE: 59 atgaagttgc
ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagt 57 <210>
SEQ ID NO 60 <211> LENGTH: 232 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic alternative sequence for hBU12 Light
Chain <400> SEQUENCE: 60 Met Lys Leu Pro Val Arg Leu Leu Val
Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser Ser Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Leu 20 25 30 Ser Pro Gly Glu Arg
Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val 35 40 45 Ser Tyr Met
His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 50 55 60 Leu
Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe 65 70
75 80 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu 85 90 95 Glu Pro Glu Asp Val Ala Val Tyr Tyr Cys Phe Gln Gly
Ser Val Tyr 100 105 110 Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys Arg Thr Val 115 120 125 Ala Ala Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys 130 135 140 Ser Gly Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155 160 Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 165 170 175 Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 180 185 190
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 195
200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210>
SEQ ID NO 61 <211> LENGTH: 19 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic alternative leader sequence for hBU12
Light Chain <400> SEQUENCE: 61 Met Lys Leu Pro Val Arg Leu
Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser Ser Ser
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 61 <210>
SEQ ID NO 1 <211> LENGTH: 19 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 leader sequence - heavy chain
region <400> SEQUENCE: 1 Met Gly Arg Leu Thr Ser Ser Phe Leu
Leu Leu Ile Val Pro Ala Tyr 1 5 10 15 Val Leu Ser <210> SEQ
ID NO 2 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH2-70/JH4 germline) - Variant HA <400> SEQUENCE: 2 Gln Val
Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro
Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 3 <211> LENGTH: 330
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain
Constant domain (IgG1) <400> SEQUENCE: 3 Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295
300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330
<210> SEQ ID NO 4 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable
Region (VH2-70/JH4 germline) - Variant HB <400> SEQUENCE: 4
Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5
10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys
Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys
Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met
Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Met Glu
Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 <210> SEQ ID NO 5 <211>
LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH2-70/JH4 germline) - Variant HC
<400> SEQUENCE: 5 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu
Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser
Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Phe
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90
95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ
ID NO 6 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH2-70/JH4 germline) - Variant HD <400> SEQUENCE: 6 Gln Val
Thr Leu Arg Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu
Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Val Leu Lys Ile Ala Ser Val Asp Pro
Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 7 <211> LENGTH: 120
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain
Variable Region (VH2-70/JH4 germline) - Variant HE
<400> SEQUENCE: 7 Gln Val Thr Leu Arg Glu Ser Gly Pro Ala Leu
Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile
Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser
Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val
Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Ala Tyr Tyr 85 90
95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> SEQ
ID NO 8 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Variable Region (Murine)
<400> SEQUENCE: 8 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile
Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser
Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile
Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile
Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys Ser
Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Asn Gln Val 65 70 75 80 Phe
Leu Lys Ile Ala Ser Val Asp Thr Ala Asp Thr Ala Ala Tyr Tyr 85 90
95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> SEQ
ID NO 9 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH4-31/JH4 germline) - Variant HF <400> SEQUENCE: 9 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 10 <211> LENGTH:
120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH4-31/JH4 germline) - Variant HG
<400> SEQUENCE: 10 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His
Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe 65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85
90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 11 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH4-31/JH4 germline) - Variant HH <400> SEQUENCE: 11 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 12 <211> LENGTH:
120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH4-31/JH4 germline) - Variant HI
<400> SEQUENCE: 12 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Phe
Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His
Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln Phe 65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85
90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 13 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Heavy Chain Variable Region
(VH4-31/JH4 germline) - Variant HJ <400> SEQUENCE: 13 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Ser Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 14 <211> LENGTH:
120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region
(VH4-31/JH4 germline) - Variant HK <400> SEQUENCE: 14 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Thr Ser 20
25 30 Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu
Glu 35 40 45 Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Val 65 70 75 80 Phe Leu Lys Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp
Ser Tyr Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr
Val Ser Ser 115 120 <210> SEQ ID NO 15 <211> LENGTH:
120 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Heavy Chain Variable Region (VH4-31/JH4 germline) - Variant HL
<400> SEQUENCE: 15 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val
Ser Gly Gly Ser Ile Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp
Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly His
Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu Lys
Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ala Tyr Tyr 85
90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210>
SEQ ID NO 16 <211> LENGTH: 22 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Light Chain Region - Leader
Sequence <400> SEQUENCE: 16 Met Asp Phe Gln Val Gln Ile Phe
Ser Phe Leu Leu Ile Ser Ala Ser 1 5 10 15 Val Ile Met Ser Arg Gly
20 <210> SEQ ID NO 17 <211> LENGTH: 107 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Variable
Region (VL-L6/Jk2 germline) - Variant LA <400> SEQUENCE: 17
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID NO 18
<211> LENGTH: 106 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain Constant domain (Kappa domain)
<400> SEQUENCE: 18 Thr Val Ala Ala Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85
90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210>
SEQ ID NO 19 <211> LENGTH: 107 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Light Chain Variable Region
(VL-L6/Jk2 germline) - Variant LB <400> SEQUENCE: 19 Glu Asn
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe Gln
Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 <210> SEQ ID NO 20 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain Variable Region (VL-L6/Jk2 germline) - Variant LC
<400> SEQUENCE: 20 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys
Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser
Gly Thr Asp His Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85
90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 21 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Variable
Region (VL-L6/Jk2 germline) - Variant LD <400> SEQUENCE: 21
Glu Asn Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp His Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys
Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID NO 22
<211> LENGTH: 107 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Light Chain Variable Region
(VL-L6/Jk2 germline) - Variant LE <400> SEQUENCE: 22 Glu Ile
Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Ser Ser Thr Ala Pro Arg Leu Leu Ile
Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Phe Gln
Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 <210> SEQ ID NO 23 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain Variable Region (VL-L6/Jk2 germline) - Variant LF
<400> SEQUENCE: 23 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys
Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser
Gly Asn Ser His Phe Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80
Asp Phe Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85
90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 24 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Light Chain Variable
Region (VL-L6/Jk2 germline) - Variant LG <400> SEQUENCE: 24
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5
10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Val Ala Val Tyr Tyr Cys
Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID NO 25
<211> LENGTH: 107 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain Variable Region (Murine) <400>
SEQUENCE: 25 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala
Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Ser Thr
Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser
Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Asn Ser
His Phe Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Val Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe
Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> SEQ ID
NO 26 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Light Chain Variable Region
(VL-A10/Jk2 germline) - Variant LH <400> SEQUENCE: 26 Glu Ile
Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20
25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
Lys 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe
Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn
Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Phe Gln
Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu
Glu Ile Lys Arg 100 105 <210> SEQ ID NO 27 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain Variable Region (VL-A10/Jk2 germline) - Variant LI
<400> SEQUENCE: 27 Glu Asn Val Leu Thr Gln Ser Pro Asp Phe
Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys
Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys 35 40 45 Asp Thr Ser Lys
Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser
Gly Thr Asp His Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85
90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 28 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Heavy Chain Variable Region (VH2-70/JH4 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(77)..(77) <223> OTHER INFORMATION: Xaa is Ser or Lys
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa is Phe or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (83)..(83) <223> OTHER INFORMATION: Xaa is Lys or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (84)..(84) <223> OTHER INFORMATION: Xaa is Ile or
Met <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (85)..(85) <223> OTHER INFORMATION: Xaa is Ala or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (86)..(86) <223> OTHER INFORMATION: Xaa is Ser or
Asn <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (87)..(87) <223> OTHER INFORMATION: Xaa is Val or
Met <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa is Ala or
Thr <400> SEQUENCE: 28 Gln Val Thr Leu Arg Glu Ser Gly Pro
Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr
Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly
Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala
His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu
Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Xaa Asn Gln Val
65 70 75 80 Xaa Leu Xaa Xaa Xaa Xaa Xaa Asp Pro Val Asp Thr Ala Xaa
Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 29 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Heavy Chain Variable Region (VH4-31/JH4 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa is Phe or Val
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa is Phe or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (29)..(29) <223> OTHER INFORMATION: Xaa is Leu or
Ile <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (73)..(73) <223> OTHER INFORMATION: Xaa is Lys or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (77)..(77) <223> OTHER INFORMATION: Xaa is Ser or
Lys <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(80) <223> OTHER INFORMATION: Xaa is Val or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa is Phe or
Ser <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa is Ala or
Val <400> SEQUENCE: 29 Gln Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr
Xaa Ser Gly Xaa Ser Xaa Ser Thr Ser 20 25 30 Gly Met Gly Val Gly
Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly
His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55 60 Leu
Lys Ser Arg Val Thr Ile Ser Xaa Asp Thr Ser Xaa Asn Gln Xaa 65 70
75 80 Xaa Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa Tyr
Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp Tyr
Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 30 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Light Chain Variable Region (VL-L6/Jk2 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is Asn or Ile
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (39)..(39) <223> OTHER INFORMATION: Xaa is Ser or
Pro <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (40)..(40) <223> OTHER INFORMATION: Xaa is Ser or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (41)..(41) <223> OTHER INFORMATION: Xaa is Thr or
Gln <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (68)..(68) <223> OTHER INFORMATION: Xaa is Asn or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (69)..(69) <223> OTHER INFORMATION: Xaa is Ser or
Asp <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa is His or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (71)..(71) <223> OTHER INFORMATION: Xaa is Phe or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (82)..(82) <223> OTHER INFORMATION: Xaa is Val or
Phe <400> SEQUENCE: 30 Glu Xaa Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln
Lys Xaa Xaa Xaa Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser
Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Xaa Xaa Xaa Xaa Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70
75 80 Asp Xaa Ala Val Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe
Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 31 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Light Chain Variable Region (VL-A10/Jk2 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is Asn or Ile
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa is His or
Phe <400> SEQUENCE: 31 Glu Xaa Val Leu Thr Gln Ser Pro Asp
Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys
Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln
Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile Lys 35 40 45 Asp Thr Ser
Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Asp Xaa Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe
Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
<210> SEQ ID NO 32 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Heavy Chain Variable Region (VH2-70/JH1-6 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(77)..(77) <223> OTHER INFORMATION: Xaa is Ser or Lys
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa is Phe or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (83)..(83) <223> OTHER INFORMATION: Xaa is Lys or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (84)..(84) <223> OTHER INFORMATION: Xaa is Ile or
Met <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (85)..(85) <223> OTHER INFORMATION: Xaa is Ala or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (86)..(86) <223> OTHER INFORMATION: Xaa is Ser or
Asn <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (87)..(87) <223> OTHER INFORMATION: Xaa is Val or
Met <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa is Ala or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (112)..(112) <223> OTHER INFORMATION: Xaa is Gln or
Arg <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (115)..(115) <223> OTHER INFORMATION: Xaa is Leu,
Thr, or Met <400> SEQUENCE: 32 Gln Val Thr Leu Arg Glu Ser
Gly Pro Ala Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr
Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly
Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp
Leu Ala His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala
50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Xaa Asn
Gln Val 65 70 75 80 Xaa Leu Xaa Xaa Xaa Xaa Xaa Asp Pro Val Asp Thr
Ala Xaa Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr
Phe Asp Tyr Trp Gly Xaa 100 105 110 Gly Thr Xaa Val Thr Val Ser Ser
115 120 <210> SEQ ID NO 33 <211> LENGTH: 120
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic hBU12 Consensus
sequence for Heavy Chain Variable Region (VH4-31/JH1-6 germline)
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (24)..(24) <223> OTHER INFORMATION: Xaa is Phe or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa is Phe or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (29)..(29) <223> OTHER INFORMATION: Xaa is Leu or
Ile <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (73)..(73) <223> OTHER INFORMATION: Xaa is Lys or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (77)..(77) <223> OTHER INFORMATION: Xaa is Ser or
Lys <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (80)..(80) <223> OTHER INFORMATION: Xaa is Val or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (81)..(81) <223> OTHER INFORMATION: Xaa is Phe or
Ser <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (94)..(94) <223> OTHER INFORMATION: Xaa is Ala or
Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (112)..(112) <223> OTHER INFORMATION: Xaa is Gln or
Arg <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (115)..(115) <223> OTHER INFORMATION: Xaa is Leu,
Thr, or Met <400> SEQUENCE: 33 Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Thr Xaa Ser Gly Xaa Ser Xaa Ser Thr Ser 20 25 30 Gly Met Gly
Val Gly Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp
Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg Tyr Asn Pro Ala 50 55
60 Leu Lys Ser Arg Val Thr Ile Ser Xaa Asp Thr Ser Xaa Asn Gln Xaa
65 70 75 80 Xaa Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Xaa
Tyr Tyr 85 90 95 Cys Ala Arg Met Glu Leu Trp Ser Tyr Tyr Phe Asp
Tyr Trp Gly Xaa 100 105 110 Gly Thr Xaa Val Thr Val Ser Ser 115 120
<210> SEQ ID NO 34 <211> LENGTH: 107 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Consensus sequence
for Light Chain Variable Region (VL-L6/Jk1-5 germline) <220>
FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:
(2)..(2) <223> OTHER INFORMATION: Xaa is Asn or Ile
<220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (39)..(39) <223> OTHER INFORMATION: Xaa is Ser or
Pro <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (40)..(40) <223> OTHER INFORMATION: Xaa is Ser or
Gly <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (41)..(41) <223> OTHER INFORMATION: Xaa is Thr or
Gln <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (68)..(68) <223> OTHER INFORMATION: Xaa is Asn or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (69)..(69) <223> OTHER INFORMATION: Xaa is Ser or
Asp <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa is His or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (71)..(71) <223> OTHER INFORMATION: Xaa is Phe or
Thr <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (82)..(82) <223> OTHER INFORMATION: Xaa is Val or
Phe <220> FEATURE: <221> NAME/KEY: MOD_RES <222>
LOCATION: (99)..(99) <223> OTHER INFORMATION: Xaa is Gln, Pro
or Gly <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (102)..(102) <223> OTHER INFORMATION:
Xaa is Lys or Arg <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (103)..(103) <223> OTHER
INFORMATION: Xaa is Leu or Val <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (104)..(104) <223>
OTHER INFORMATION: Xaa is Glu or Asp <400> SEQUENCE: 34 Glu
Xaa Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val Ser Tyr Met
20 25 30 His Trp Tyr Gln Gln Lys Xaa Xaa Xaa Ala Pro Arg Leu Leu
Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg
Phe Ser Gly Ser 50 55 60 Gly Ser Gly Xaa Xaa Xaa Xaa Leu Thr Ile
Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Xaa Ala Val Tyr Tyr Cys Phe
Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe Gly Xaa Gly Thr Xaa
Xaa Xaa Ile Lys Arg 100 105 <210> SEQ ID NO 35 <211>
LENGTH: 107 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Consensus sequence for Light Chain Variable Region (VL-A10/Jk1-5
germline) <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (2)..(2) <223> OTHER INFORMATION: Xaa
is Asn or Ile <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (70)..(70) <223> OTHER INFORMATION: Xaa
is His or Phe <220> FEATURE: <221> NAME/KEY: MOD_RES
<222> LOCATION: (99)..(99) <223> OTHER INFORMATION: Xaa
is Gln, Pro or Gly <220> FEATURE: <221> NAME/KEY:
MOD_RES <222> LOCATION: (102)..(102) <223> OTHER
INFORMATION: Xaa is Lys or Arg <220> FEATURE: <221>
NAME/KEY: MOD_RES <222> LOCATION: (103)..(103) <223>
OTHER INFORMATION: Xaa is Leu or Val <220> FEATURE:
<221> NAME/KEY: MOD_RES <222> LOCATION: (104)..(104)
<223> OTHER INFORMATION: Xaa is Glu or Asp <400>
SEQUENCE: 35 Glu Xaa Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val
Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser
Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Asp Gln
Ser Pro Lys Leu Leu Ile Lys 35 40 45 Asp Thr Ser Lys Leu Ala Ser
Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp
Xaa Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu 65 70 75 80 Asp Ala Ala
Thr Tyr Tyr Cys Phe Gln Gly Ser Val Tyr Pro Phe Thr 85 90 95 Phe
Gly Xaa Gly Thr Xaa Xaa Xaa Ile Lys Arg 100 105 <210> SEQ ID
NO 36 <211> LENGTH: 326 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Constant Domain (IgG2)
<400> SEQUENCE: 36 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155
160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln
Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr
Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr
Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280
285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> SEQ
ID NO 37 <211> LENGTH: 290 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Constant Domain (IgG3)
<400> SEQUENCE: 37 Gln Met Gln Gly Val Asn Cys Thr Val Ser
Ser Glu Leu Lys Thr Pro 1 5 10 15 Leu Gly Asp Thr Thr His Thr Cys
Pro Arg Cys Pro Glu Pro Lys Ser 20 25 30 Cys Asp Thr Pro Pro Pro
Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys 35 40 45 Asp Thr Pro Pro
Pro Cys Pro Arg Cys Pro Glu Pro Lys Ser Cys Asp 50 55 60 Thr Pro
Pro Pro Cys Pro Arg Cys Pro Ala Pro Glu Leu Leu Gly Gly 65 70 75 80
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 85
90 95 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu 100 105 110 Asp Pro Glu Val Gln Phe Lys Trp Tyr Val Asp Gly Val
Gln Val His 115 120 125 Asn Ala Lys Thr Lys Pro Arg Glu Gln Gln Phe
Asn Ser Thr Phe Arg 130 135 140 Val Val Ser Val Leu Thr Val Leu His
Gln Asn Trp Leu Asp Gly Lys 145 150 155 160 Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 165 170 175 Lys Thr Ile Ser
Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 180 185 190 Thr Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 195 200 205
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 210
215 220 Glu Ser Ser Gly Gln Pro Glu Asn Asn Tyr Asn Thr Thr Pro Pro
Met 225 230 235 240 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp 245 250 255 Lys Ser Arg Trp Gln Gln Gly Asn Ile Phe
Ser Cys Ser Val Met His 260 265 270 Glu Ala Leu His Asn Arg Phe Thr
Gln Lys Ser Leu Ser Leu Ser Pro 275 280 285 Gly Lys 290 <210>
SEQ ID NO 38 <211> LENGTH: 327 <212> TYPE: PRT
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic hBU12 Heavy Chain Constant Domain
(IgG4) <400> SEQUENCE: 38 Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70
75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys
Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195
200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val
Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315
320 Leu Ser Leu Ser Leu Gly Lys 325 <210> SEQ ID NO 39
<211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Heavy Chain Constant Domain Variant (IgG1V1)
<400> SEQUENCE: 39 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85
90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110 Pro Ala Pro Pro Val Ala Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210
215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> SEQ ID NO
40 <211> LENGTH: 57 <212> TYPE: DNA <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic hBU12 Heavy Chain Region - Leader Sequence
<400> SEQUENCE: 40 atgggcaggc ttacttcttc attcttgttg
ctgattgtcc ctgcatatgt cctgtcc 57 <210> SEQ ID NO 41
<211> LENGTH: 360 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Heavy Chain Variable Region (VH4-31/JH4 germline) -
Variant HF <400> SEQUENCE: 41 caggttcagc tgcaagagtc
tggccctggg ttggttaagc cctcccagac cctcagtctg 60 acttgtactg
tgtctggggg ttcaatcagc acttctggta tgggtgtagg ctggattagg 120
cagcacccag ggaagggtct ggagtggatt ggacacattt ggtgggatga tgacaagaga
180 tataacccag ccctgaagag cagagtgaca atctctgtgg atacctccaa
gaaccagttt 240 agcctcaagc tgtccagtgt gacagctgca gatactgctg
tctactactg tgctagaatg 300 gaactttggt cctactattt tgactactgg
ggccaaggca cccttgtcac agtctcctca 360 <210> SEQ ID NO 42
<211> LENGTH: 993 <212> TYPE: DNA <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Heavy Chain Constant domain (IgG1) <400>
SEQUENCE: 42 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag
cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc
ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg
cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag
cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca
acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga
360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc
ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc
ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc
aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag
cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt
gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag
720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc
cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact
acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac
agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc
atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc
tctccctgtc tccgggtaaa tga 993 <210> SEQ ID NO 43 <211>
LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain Region - Leader Sequence <400> SEQUENCE: 43
atggattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc
60 agagga 66 <210> SEQ ID NO 44 <211> LENGTH: 321
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic hBU12 Light Chain
Variable Region (VL-L6/Jk2 germline) - Variant LG <400>
SEQUENCE: 44 gaaattgttc tcacccagtc tccagcaacc ctgtctctct ctccagggga
aagggctacc 60 ctgagctgca gtgccagctc aagtgtaagt tacatgcact
ggtaccagca gaagccaggg 120 caggctccca gactcctgat ttatgacaca
tccaaactgg cttctggtat tccagcaagg 180 ttcagtggca gtgggtctgg
aacagatttt acactcacaa tcagcagcct ggagccagag 240 gatgttgctg
tctattactg ttttcagggg agtgtatacc cattcacttt tggccaaggg 300
acaaagttgg aaatcaaaag a 321 <210> SEQ ID NO 45 <211>
LENGTH: 321 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain Constant domain (Kappa domain) <400> SEQUENCE: 45
actgtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga
60 actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa
agtacagtgg 120 aaggtggata acgccctcca atcgggtaac tcccaggaga
gtgtcacaga gcaggacagc 180 aaggacagca cctacagcct cagcagcacc
ctgacgctga gcaaagcaga ctacgagaaa 240 cacaaagtct acgcctgcga
agtcacccat cagggcctga gctcgcccgt cacaaagagc 300 ttcaacaggg
gagagtgtta g 321 <210> SEQ ID NO 46 <211> LENGTH: 7
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain
CDR1 <400> SEQUENCE: 46 Thr Ser Gly Met Gly Val Gly 1 5
<210> SEQ ID NO 47 <211> LENGTH: 16 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic hBU12 Heavy Chain CDR2
<400> SEQUENCE: 47 His Ile Trp Trp Asp Asp Asp Lys Arg Tyr
Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> SEQ ID NO 48
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Heavy Chain CDR3 <400> SEQUENCE: 48 Met Glu
Leu Trp Ser Tyr Tyr Phe Asp Tyr 1 5 10 <210> SEQ ID NO 49
<211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain CDR1 <400> SEQUENCE: 49 Ser Ala
Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> SEQ ID NO 50
<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic hBU12 Light Chain CDR2 <400> SEQUENCE: 50 Asp Thr
Ser Lys Leu Ala Ser 1 5 <210> SEQ ID NO 51 <211>
LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic hBU12
Light Chain CDR3 <400> SEQUENCE: 51 Phe Gln Gly Ser Val Tyr
Pro Phe Thr 1 5 <210> SEQ ID NO 52
<211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:
Artificial <220> FEATURE: <223> OTHER INFORMATION:
Synthetic linker <400> SEQUENCE: 52 Gly Phe Leu Gly 1
<210> SEQ ID NO 53 <211> LENGTH: 1410 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic alternative sequence for
hBU12 Heavy Chain <400> SEQUENCE: 53 atgggatgga gctggatctt
tcttttcctc ctgtcaggaa ctgcaggtgt ccattgtcag 60 gttcagctgc
aagagtctgg ccctgggttg gttaagccct cccagaccct cagtctgact 120
tgtactgtgt ctgggggttc aatcagcact tctggtatgg gtgtaggctg gattaggcag
180 cacccaggga agggtctgga gtggattgga cacatttggt gggatgatga
caagagatat 240 aacccagccc tgaagagcag agtgacaatc tctgtggata
cctccaagaa ccagtttagc 300 ctcaagctgt ccagtgtgac agctgcagat
actgctgtct actactgtgc tagaatggaa 360 ctttggtcct actattttga
ctactggggc caaggcaccc ttgtcacagt ctcctcagct 420 agcaccaagg
gcccatctgt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480
acagctgccc tgggctgcct ggtcaaggac tacttccctg aacctgtgac agtgtcctgg
540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca
gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca
gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac
accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac
atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc
tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac
900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca
gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg
actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc
ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga
accacaggtg tacaccctgc ccccatcccg ggatgagctg 1140 accaagaacc
aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg
1260 gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag
caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc
tgcacaacca ctacacacag 1380 aagagcctct ccctgtctcc gggtaaatga 1410
<210> SEQ ID NO 54 <211> LENGTH: 57 <212> TYPE:
DNA <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic alternative leader
sequence for hBU12 Heavy Chain <400> SEQUENCE: 54 atgggatgga
gctggatctt tcttttcctc ctgtcaggaa ctgcaggtgt ccattgt 57 <210>
SEQ ID NO 55 <211> LENGTH: 993 <212> TYPE: DNA
<213> ORGANISM: Artificial <220> FEATURE: <223>
OTHER INFORMATION: Synthetic alternative sequence for hBU12 Heavy
Chain - Constant domain <400> SEQUENCE: 55 gctagcacca
agggcccatc tgtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagctg ccctgggctg cctggtcaag gactacttcc ctgaacctgt gacagtgtcc
120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct
acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca
gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc
aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca
cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct
tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg
480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga
gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc
aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc
ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg
agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga
accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa
gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacaca 960 cagaagagcc tctccctgtc tccgggtaaa tga 993
<210> SEQ ID NO 56 <211> LENGTH: 469 <212> TYPE:
PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic alternative sequence for
hBU12 Heavy Chain <400> SEQUENCE: 56 Met Gly Trp Ser Trp Ile
Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Cys Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser
Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45
Ser Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln His Pro Gly Lys 50
55 60 Gly Leu Glu Trp Ile Gly His Ile Trp Trp Asp Asp Asp Lys Arg
Tyr 65 70 75 80 Asn Pro Ala Leu Lys Ser Arg Val Thr Ile Ser Val Asp
Thr Ser Lys 85 90 95 Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ala Arg Met Glu Leu
Trp Ser Tyr Tyr Phe Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180
185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val 210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305
310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425
430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 450 455 460 Leu Ser Pro Gly Lys 465 <210> SEQ ID NO
57 <211> LENGTH: 19 <212> TYPE: PRT <213>
ORGANISM: Artificial <220> FEATURE: <223> OTHER
INFORMATION: Synthetic alternative leader sequence for hBU12 Heavy
Chain <400> SEQUENCE: 57 Met Gly Trp Ser Trp Ile Phe Leu Phe
Leu Leu Ser Gly Thr Ala Gly 1 5 10 15 Val His Cys <210> SEQ
ID NO 58 <211> LENGTH: 699
<212> TYPE: DNA <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic alternative
sequence for hBU12 Light Chain <400> SEQUENCE: 58 atgaagttgc
ctgttaggct gttggtgctg atgttctgga ttcctgcttc cagcagtgaa 60
attgttctca cccagtctcc agcaaccctg tctctctctc caggggaaag ggctaccctg
120 agctgcagtg ccagctcaag tgtaagttac atgcactggt accagcagaa
gccagggcag 180 gctcccagac tcctgattta tgacacatcc aaactggctt
ctggtattcc agcaaggttc 240 agtggcagtg ggtctggaac agattttaca
ctcacaatca gcagcctgga gccagaggat 300 gttgctgtct attactgttt
tcaggggagt gtatacccat tcacttttgg ccaagggaca 360 aagttggaaa
tcaaaagaac tgtggctgca ccatctgtct tcatcttccc gccatctgat 420
gagcagttga aatctggaac tgcctctgtt gtgtgcctgc tgaataactt ctatcccaga
480 gaggccaaag tacagtggaa ggtggataac gccctccaat cgggtaactc
ccaggagagt 540 gtcacagagc aggacagcaa ggacagcacc tacagcctca
gcagcaccct gacgctgagc 600 aaagcagact acgagaaaca caaagtctac
gcctgcgaag tcacccatca gggcctgagc 660 tcgcccgtca caaagagctt
caacagggga gagtgttag 699 <210> SEQ ID NO 59 <211>
LENGTH: 57 <212> TYPE: DNA <213> ORGANISM: Artificial
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
alternative leader sequence for hBU12 Light Chain <400>
SEQUENCE: 59 atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc
cagcagt 57 <210> SEQ ID NO 60 <211> LENGTH: 232
<212> TYPE: PRT <213> ORGANISM: Artificial <220>
FEATURE: <223> OTHER INFORMATION: Synthetic alternative
sequence for hBU12 Light Chain <400> SEQUENCE: 60 Met Lys Leu
Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser
Ser Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu 20 25
30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser Val
35 40 45 Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu 50 55 60 Leu Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Ile
Pro Ala Arg Phe 65 70 75 80 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu 85 90 95 Glu Pro Glu Asp Val Ala Val Tyr
Tyr Cys Phe Gln Gly Ser Val Tyr 100 105 110 Pro Phe Thr Phe Gly Gln
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 115 120 125 Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 130 135 140 Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 145 150 155
160 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
165 170 175 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser 180 185 190 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys 195 200 205 Val Tyr Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr 210 215 220 Lys Ser Phe Asn Arg Gly Glu Cys
225 230 <210> SEQ ID NO 61 <211> LENGTH: 19 <212>
TYPE: PRT <213> ORGANISM: Artificial <220> FEATURE:
<223> OTHER INFORMATION: Synthetic alternative leader
sequence for hBU12 Light Chain <400> SEQUENCE: 61 Met Lys Leu
Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala 1 5 10 15 Ser
Ser Ser
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